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Effects of fatty acids on human neutrophil-mediated killing of Plasmodium falciparum asexual blood forms were investigated by using a quantitative radiometric assay. The results showed that the antiparasitic activity of neutrophils can be greatly increased (>threefold) by short-term treatment with fatty acids with 20 to 24 carbon atoms and at least three double bonds. In particular, the n-3 polyenoic fatty acids, eicosapentaenoic and docosahexaenoic acids, and the n-6 fatty acid, arachidonic acid, significantly enhanced neutrophil antiparasitic activity. This effect was >1.5-fold higher than that induced by an optical concentration of the known agonist cytokine tumor necrosis factor alpha (TNF-alpha). At suboptimal concentrations, the combination of arachidonic acid and TNF-alpha caused a synergistic increase in neutrophil-mediated parasite killing. The fatty acid-induced effect was independent of the availability of serum opsonins but dependent on the structure of the fatty acids. The length of the carbon chain, degree of unsaturation, and availability of a free carboxyl group were important determinants of fatty acid activity. The fatty acids which increased neutrophil-mediated killing primed the enhanced superoxide radical generation of neutrophils in response to P. falciparum as detected by chemiluminescence. Scavengers of oxygen radicals significantly reduced the fatty acid-enhanced parasite killing, but cyclooxygenase and lipoxygenase inhibitors had no effect. These findings have identified a new class of immunoenhancers that could be exploited to increase resistance against Plasmodium species.

As chemoresistance of Plasmodium falciparum to chloroquine has arisen, new ways of combating the infection are needed. Similarities exist between the multidrug resistance of mammalian cells and chloroquine resistance of P. falciparum, based on the occurrence of internucleosomal deoxyribonucleic acid (DNA) breakdown and the ability of some anticancer drugs and chloroquine to induce apoptosis. Using chloroquine, oligonucleosomal DNA fragmentation was observed with a sensitive strain of P. falciparum, but not with a resistant one. This suggests that apoptosis may be involved in the action of chloroquine on the parasite.

Catalytic RNA (ribozymes) suppressed the growth of the human malarial parasite Plasmodium falciparum in vitro. The phosphorothioated hammerhead ribozymes targeted unique regions of the P. falciparum carbamoyl-phosphate synthetase II gene. The P. falciparum carbamoyl-phosphate synthetase II gene encodes the first and limiting enzyme in the pathway, and its mRNA transcript contains two large insert regions absent in other carbamoyl-phosphate synthetases, including that from humans. These inserts are ideal targets for nucleic acid therapy. Exogenous delivery of ribozymes to cultures reduced malarial viability up to 55% at 0.5 microM ribozyme concentrations, which is significantly greater than control levels (5-15% reduction), suggesting a sequence-specific inhibition. This inhibition was shown to be stage-specific, with optimal inhibitions being detected after 24 h, coincident with maximal production of the carbamoyl-phosphate synthetase enzyme in the course of the life cycle of the parasite. A decrease in total carbamoyl-phosphate synthetase activity was observed only in cultures treated with the ribozymes. The task of developing alternative therapeutic agents against malaria is urgent due to the evolution of drug-resistant strains of P. falciparum, the most virulent of all human malarial parasites. Another critical issue to be addressed is the possibility of eliminating or reducing any systemic toxicity to the host, which can potentially be provided by nucleic acid therapy. This work is the first reported assessment of the ability of ribozymes as antimalarials. Ribozyme inhibition assays can also aid in identifying important antimalarial loci for chemotherapy. The malarial parasite can, in turn, be a useful in vivo host to study the catalysis and function of new ribozyme designs.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha-tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.

A method for determining the infectious reservoir of malaria (K) and vector survival rate (P) by measuring oocyst size and discriminating between the most recent and other infections is described. In the laboratory the mean diameter of 3 d oocysts in Anopheles gambiae, kept at 26 degrees C, was 11.5 microns and the mean diameter at day 5 was 24.5 microns. Oocyst sizes in wild caught mosquitoes from southern Tanzania, that had fed on the occupants of bed nets with holes in the sides, were more variable. 2060 A. gambiae s.l. and 1982 A. funestus were examined for oocysts 3 d after feeding; 796 and 654 oocysts from the 153 and 170 infected females, respectively, were measured. Because of misclassification errors, the use of a simple cut-off model, in which all oocysts less than 17.5 microns in diameter were considered to have arisen from the most recent feed, was thought to overestimate K and underestimate P. A statistical model which allows for overlap in the oocyst size distributions is described. Estimates of the infectious reservoir derived from this model were 2.8% for A. gambiae s.l. and 4.2% for A. funestus, and the estimated survival rates per gonotrophic cycle were 65.5% and 52.9%, respectively. The utility of measuring oocyst size in naturally infected mosquitoes is discussed.

X-ray microscopes provide higher resolution than visible light microscopes. Wet, biological materials with a water thickness of up to about 10 microns can be imaged with good contrast using soft X-rays with wavelengths between the oxygen and carbon absorption edges (at 24 and 43 A). The Stony Brook group has developed and operates a scanning transmission X-ray microscope (STXM) at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The microscope is used for imaging with a current resolution of 50 nm, and for elemental and chemical state mapping. Radiation damage imposes a significant limitation upon high resolution X-ray microscopy of room temperature wet specimens. Experience from electron microscopy suggests that cryo techniques allow vitrified specimens to be imaged repeatedly. This is due to the increased radiation stability of biological specimens in the frozen hydrated state. Better radiation stability has been shown recently with a cryo transmission X-ray microscope developed by the University of Göttingen, operating at the BESSY storage ring in Berlin, Germany. At Stony Brook, we are developing a cryo scanning transmission X-ray microscope (CryoSTXM) to carry out imaging and spectro-microscopy experiments on frozen hydrated specimens. This article will give an outlook onto the research projects that we plan to perform using the CryoSTXM.

Infection of human erythrocytes with the malaria parasite, Plasmodium falciparum, results in the exposure of amino acid residues 542-555 of the anion-exchange protein, band 3, in a conformation that enables the cell to adhere to C32 amelanotic melanoma cells. Attempts to isolate this adhesive form from infected cells by immunoaffinity were unsuccessful, and so other approaches were utilized. Chinese hamster ovary (CHO) cells transfected with cDNA encoding the first 578 amino acid residues of human band 3 protein transiently expressed the protein efficiently. A murine monoclonal antibody (MAb) that specifically recognizes the adhesin exposed on the surface of erythrocytes bearing mature stages of P. falciparum immunostained some transfected cells, confirming that the first 578 amino residues are sufficient for the adhesive conformation. As a more efficient alternative to transgenic expression of the adhesin, microspheres with covalently bound peptides fashioned on band 3 sequences previously found to be adherent (residues 546-553 and 820-829 and called pfalhesin) were produced. The pfalhesin-coated microspheres specifically bound to C32 amelanotic melanoma cells, whereas microspheres coupled with a scrambled version of residues 546-553 had little binding capacity for melanoma cells. These results demonstrate that the previously identified band 3-related peptides that inhibit cytoadherence interact directly with target cells and suggest that microspheres with covalently coupled peptides might constitute novel 'artificial' P. falciparum-infected erythrocytes for use in in vitro and in vivo studies.

Trifluralin, a herbicide which is known to bind to plant and algal tubulin, induced ultrastructural changes in the microtubules of the mature Plasmodium falciparum gametocytes in vitro. Trifluralin treatment led to disassembly of the well ordered subpellicular microtubules, whereas it had no effect on microtubules of human platelets or of rat neuronal cells in vitro. The disassembled microtubules showed fragmented large tubular structures, which frequently were associated with the pellicular membranes. Electron microscopic autoradiography showed radioactive trifluralin associated with the microtubule fragments. These results provide evidence that trifluralin selectively binds to microtubules in malaria parasites and causes disruption of their structure.

Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. The sequences of full-length clones of the large and small RNR subunits revealed an open reading frame encoding 806 and 349 amino acids, respectively, and showed significant identity with other RNR sequences in the data base. RNA blot analysis showed that the size of the large and small RNR subunit transcripts are 5.4 kb and 2.2 kb, respectively. Both the RNR subunit transcripts fluctuate in level during the cell cycle, reaching a peak preceding maximal DNA synthesis activity. An oligodeoxynucleotide phosphorothioate that is complementary to sequences around the translational initiation codon of the small RNR subunit showed significant inhibition of growth, as measured by the inhibition in DNA synthesis.

Berenil, which is known to inhibit synthesis of nucleic acid, DNA and RNA, and polyamine, induced gametocytogenesis in cultured Plasmodium falciparum, although the mechanism by which Berenil induces gametocytogenesis is unknown. Asexual parasites of a strain of P. falciparum that seldom produce gametocytes in in vitro culture began gametocytogenesis after 2-24 hr treatment with RPMI 1640 medium containing Berenil (final concentration, 0.1-10.0 micrograms/ml) with horse serum. Gametocytogenesis was consistently observed from 3 days after the addition of Berenil to culture. After parasites were cultivated in RPMI 1640 medium containing Berenil (final concentration, 3.0-10.0 micrograms/ml) and concanavalin A with horse serum for 2 hr, Berenil and concanavalin A were removed from culture medium by centrifugation. Nevertheless, induction of gametocytogenesis was observed. The addition of concanavalin A (final concentration, 10 micrograms/ml) to RPMI 1640 medium containing Berenil enhanced the induction of gametocytogenesis by Berenil. However, when RPMI 1640 medium with concanavalin A was used without the addition of Berenil, no gametocytogenesis was observed as in RPMI 1640 medium alone.

Stable transformants of Chinese hamster ovary (CHO) cell lines expressing high levels of human CD36 or intercellular adhesion molecule-1 (ICAM-1) have been produced as target cells for cytoadherence of Plasmodium falciparum-infected erythrocytes. An improved adherence microassay has been designed using small sample volumes and allowing convenient and reliable measurements on a large number of samples. The assay can be used both with purified proteins spotted on plastic and with the stably transformed CHO cell lines. The same assay plate can be evaluated either microscopically or by scintillation counting after use of 3H-hypoxanthine-labeled parasites. Using the microassay, functional expression of the transfected receptor molecules on CHO-CD36 and CHO-ICAM was confirmed using parasites with different cytoadherence phenotypes and cytoadherence inhibition experiments with a panel of anti-CD36 antibodies. The use of isolates from The Gambia confirmed the applicability of these assays for laboratory studies of these isolates.

The sporogonic cycle of Plasmodium falciparum was obtained in vitro. Mature gametocytes, from blood-stage cultures, produced gametes that underwent fertilization at elevated pH and ambient temperatures. Wheat germ agglutinin stimulated transformation of zygotes into retorts and ookinetes. Twenty-four hours thereafter, 18-49% mature ookinetes and 10-20% intermediate retort forms were counted. Cultures were seeded onto a basement membrane-like gel (Matrigel) in coculture with Drosophila melanogaster cells. Both ookinetes and retorts attached to Matrigel and transformed into oocysts. Mature oocysts and sporozoites expressed circumsporozoite protein. The entire life cycle of Plasmodium falciparum, the most important malaria pathogen of humans, can now be studied in vitro.

Several components were tested for their ability to replace human serum in cultures of Plasmodium falciparum set up for the development of gametocytes. Besides a serum-free medium, A*I*M*V (Gibco BRL), RPMI medium supplemented with commercially available serum substitutes was used to culture gametocytes. The following substances served as serum replacements: Basal Medium Supplement (Biochrom), Ultroser G (Gibco BRL) and Nutridoma-SR (Boehringer Mannheim). All serum-free additives supported some parasite growth, but only in RPMI supplemented with Nutridoma-SR were morphologically mature gametocytes obtained. The asexual forms developed almost as well as in RPMI with human serum added.

Sequestration of Plasmodium falciparum-infected erythrocytes to the capillary endothelium can cause obstruction and localized tissue damage. Occlusion of vessels in falciparum malaria infection has been related to two properties of the parasite: adhesion to endothelial cells and rosette formation. Our study on P. falciparum isolates from Thailand producing variable numbers of rosettes suggests the involvement of rosettes in capillary blockage caused by direct adhesion of the rosette-forming infected erythrocytes to various target cells, e.g., live human umbilical vein endothelial cells, monocytes, and platelets. These rosettes did not bind Formalin-fixed target cells, nor did they bind to live or fixed C32 or G361 melanoma cells. Classification of the receptors involved in cytoadherence of endothelial cells and monocytes by specific antibody blocking and flow cytometry indicated that CD36 was involved in the adherence of monocytes but that other receptors besides CD36 may be involved in parasite adherence to endothelial cells. The cytoadherence of infected erythrocytes to monocytes was also associated with CD54 (ICAM-1). Further, differentiation of adherent monocytes resulted in an inversion of CD36 and CD54 levels on the cell surface which correlated with a decrease in surface binding of infected erythrocytes. This observation suggests that the state of cell activation and differentiation may also contribute to sequestration of parasites and to the pathogenesis of malaria.

Two gametocyte-forming clones, HB-3 and 3D7, were used. Concentrates of late stage parasites were mixed with bloods containing different proportions of young erythrocytes, and the parasitemia and proportion of gametocytes determined after 2, 3 or 4 days of culture. Significantly more gametocytes were formed in light cells than in heavy cells separated from the same normal blood samples. Up to seven times more gametocytes were formed in reticulocyte-rich bloods from patients with sickle cell anemia than in normal control blood.

We previously showed that the Uganda Palo Alto line of Plasmodium falciparum propagated in Saimiri monkeys and the line maintained in culture in human erythrocytes for many years in our laboratory are genetically unrelated (T. Fandeur, S. Bonnefoy, and O. Mercereau-Puijalon, Mol. Biochem. Parasitol. 47:167, 1991). When injected into a splenectomized Saimiri monkey, the in vitro-derived Palo Alto population procured a long-lasting, low-grade parasitemia that was spontaneously resolved by the animal. This line was propagated by serial blood transfers in two other monkeys without enhancement of the virulence of the parasites. The genetic characteristics of parasite samples corresponding to the different passages of the line in monkeys were stable for the several markers examined (pPF11.1, MSA1, and MSA2), although microheterogeneity was detected in telomeric and subtelomeric regions of chromosomes. Interestingly, in vitro-derived Palo Alto parasites induced a strong, potent immunity that enabled the monkeys to completely block subsequent challenge with two different heterologous lethal P. falciparum lines. These attenuated parasites are thus genetically stable in monkeys and represent an attractive model for assessing the feasibility of a live attenuated malaria vaccine.

Merozoites of the malaria parasite Plasmodium falciparum possess on their surface proteolytically processed fragments of the merozoite surface protein-1 (MSP1). Secondary processing of one of these fragments, MSP1(42), always occurs prior to, or at the point of successful erythrocyte reinvasion. It is shown that a product of this secondary processing, MSP1(33), is shed in the form of a noncovalently-associated complex with a number of other proteins, including the MSP1-derived species MSP1(38) and MSP1(83). Secondary processing of MSP1(42) is inhibited by the chelating agents ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethyl ether)-tetraacetic acid (EGTA), and this inhibition is reversible by addition of excess calcium. Secondary processing occurs in preparations of washed, disrupted merozoites, and is inhibited by the protease inhibitors phenylmethylsulphonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), indicating that the protease responsible is a membrane-associated serine protease.

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.

The DNA polymerase-alpha of Plasmodium falciparum was characterized according to aphidicolin sensitivity and immunological reactivity with monoclonal anti-sera against human DNA polymerase-alpha. Two major (105 and 72 kDa) and two minor (180 and 130 kDa) catalytic subunits of P. falciparum DNA polymerase-alpha were detected on activity gels. Activity gels did not indicate the presence of a DNA polymerase-beta in P. falciparum. Metabolically labeled polypeptides at 180, 105, 72, and 52 kDa were immunoprecipitated from Plasmodium nuclear extracts with the anti-KB cell DNA polymerase-alpha monoclonal antibody and, by size, correspond to the major subunits of mammalian DNA polymerase-alpha. The monoclonal antibody also neutralized Plasmodium DNA polymerase activity. Plasmodium DNA polymerase was synthesized predominantly at an early schizont stage at which time the parasite began to synthesize its DNA and multiply. No evidence for phosphorylation of the major catalytic subunit was obtained. Plasmodium growth, DNA synthesis, and DNA polymerase activity were inhibited significantly in parallel by aphidicolin. These results suggest that P. falciparum has a typical eukaryotic DNA polymerase-alpha and that regulation of its activity appears to be at the transcriptional level.

Deferoxamine (DF) has antimalarial activity that can be demonstrated in vitro and in vivo. This study is designed to examine the speed of onset and stage dependency of growth inhibition by DF and to determine whether its antimalarial activity is cytostatic or cytocidal. Growth inhibition was assessed by suppression of hypoxanthine incorporation and differences in morphologic appearance between treated and control parasites. Using synchronized in vitro cultures of Plasmodium falciparum, growth inhibition by DF was detected within a single parasite cycle. Ring and nonpigmented trophozoite stages were sensitive to the inhibitory effect of DF but cytostatic antimalarial activity was suggested by evidence of parasite recovery in later cycles. However, profound growth inhibition, with no evidence of subsequent recovery, occurred when pigmented trophozoites and early schizonts were exposed to DF. At this stage in parasite development, the activity of DF was cytocidal and furthermore, the critical period of exposure may be as short as 6 hours. These observations suggest that iron chelators may have a role in the treatment of clinical malaria.

The growth of the malarial parasite P. falciparum was arrested by the adenine containing nucleoside sinefungin at the trophozoite stage. The synthesis of DNA, of polyamines and the specific proteins of the schizont stage were completely blocked by the drug. The inhibition of DNA synthesis was not due to a decrease in the amount of DNA polymerase, but to the depletion of polyamines which are required for DNA synthesis.

Sera of negroes of African origin and of indians, living in a malaria endemic village on the Pacific Coast of Colombia, were analyzed to see if they could block intraerythrocytic Plasmodium falciparum growth in vitro. A group of mestizos from a malaria-free city in Colombia was used as a negative control. Blood of each individual was studied for the presence of circulating parasites by thick and thin smears and their sera for antimalarial antibodies by IFAT and IRMA techniques. The inhibition of the intraerythrocytic growth induced by these sera was assessed by [3H]Hypoxanthine incorporation. All groups showed inhibitory activity independent of their exposure to malaria. Negro sera had the highest inhibitory activity even following the removal of antibody, and also the highest antimalarial antibody titers. The group of indians had reduced inhibitory activity and lower antibody titers compared to the negro sera. In the group of mestizos, who reported no malaria exposure, 14% had antibodies to asexual blood forms of P. falciparum and 60% induced significant inhibition.

We have examined immune responses to a cultured Plasmodium falciparum gametocyte lysate and to an affinity-purified preparation of the 48/45-kDa gamete surface Ag in a group of 30 malaria immune individuals and in 24 Europeans with no previous exposure to malaria. Cellular responses were assessed in vitro by lymphoproliferation and production of IFN-gamma; antigamete antibodies were detected by immunofluorescence, Western blotting, and competitive ELISA. Cells from all the malaria immune donors responded to the gametocyte lysate in both assays while cells from nonimmune donors gave only weak proliferative responses. Antigamete antibodies were detected in the serum of all the immune donors but not in serum from nonimmunes. Nonimmune donors were completely unresponsive to the purified 48/45-kDa surface Ag while cells from 40% of immune donors responded by either proliferation or IFN-gamma production. Only 3 of 30 immune donors had detectable antibodies to the 48/45-kDa Ag. Class II HLA type was determined for 27 of the immune donors but no relationship between HLA-DR or -DQ and responsiveness to the 48/45-kDa Ag was discerned. The possible reasons for limited recognition of this gamete surface Ag are discussed.

The effects of serum and human lipoproteins (HDL, VLDL, LDL) were investigated on the intraerythrocytic cycle of P. falciparum using in vitro synchronized cultures. The reinvasion process of erythrocytes by merozoites and the development until the young trophozoite stage are independent of serum components. In the absence of serum, schizogony did not occur. However, addition of serum before the 24th hour of culture in basal medium restores a normal schizogony. Serum replacement by the different lipoprotein fractions showed that only the HDL fraction was able to assure a complete schizogony as well as a normal erythrocyte reinvasion. No division was observed with the apolipoproteins.

Clones of Plasmodium falciparum prepared from recent isolates of infected blood were studied to determine the molecular mechanism of naturally occurring pyrimethamine resistance. Total DNA, as well as thymidylate synthetase and dihydrofolate reductase activities, were characterized from these lines. Restriction analysis of DNA from pyrimethamine-susceptible and -resistant lines of the parasite showed no obvious amplification of any DNA fragment. Further, analysis of DNA from resistant and susceptible lines by centrifugation in cesium chloride-ethidium bromide revealed no extrachromosomal amplification in the resistant line. Comparison of the dihydrofolate reductase enzyme activity in the two lines revealed similar KmS for substrate but a large difference in the inhibition constant for pyrimethamine. Additionally, the enzyme from the resistant line was considerably more stable in vitro than the corresponding enzyme from the susceptible line. The thymidylate synthetase activity in the two lines was similar and unaffected by pyrimethamine. The mechanism of drug resistance in this isolate involves altered properties of the dihydrofolate reductase conferring both a different affinity for the drug and increased stability.

Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.

A man with a carcinoma presented an unusual cytology P. falciparum transfusion malaria. The authors suspect cytotoxic chemotherapy for having induced these cytological changes.

The effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum grown in vitro was examined over a wide range of concentrations. Cyclic AMP inhibited both processes in a stage-specific manner. Asexual maturation was inhibited from shortly after parasite entry into the red cell through the ring stage. However, trophozoites and schizonts matured normally in the presence of cyclic AMP and produced infectious merozoites. Gametocyte formation was inhibited by 95% when 1.0 mM cyclic AMP was added to synchronously growing parasites in the ring stage of development but was only inhibited by 15% when added in the trophozoite or schizont stages. Cyclic AMP was not found to increase gametocyte formation over a wide range of concentrations.

The metabolism of phospholipids in synchronous Plasmodium falciparum-infected erythrocytes was studied over one cycle of 48 h by the incorporation of labeled palmitate, serine, choline, and myo-inositol into cellular lipids. The rates of incorporation of palmitate and serine into total phospholipids and of choline into phosphatidylcholine (PC) were linear with the maturation of the parasite, increasing by a factor of 2-5.6 according to the precursors. The rate of inositol incorporation into phosphatidylinositol was 9.6 times higher at the schizont stage than at the ring stage, with a marked increase in the second half of the cycle. A significant incorporation of palmitate into triglycerides also occurred during the schizont stage of the parasite. The incorporations of serine and palmitate into phosphatidylethanolamine (PE) and PC showed a net increase at approximately the twentieth hour of the cycle, while the radioactivities recovered in phosphatidylserine (PS) had already reached a maximum by this time. These findings indicate an instantaneous transformation of PS into PE and PC through a decarboxylation of PS into PE, then a methylation of PE into PC during the second half of the cycle. Although PS is a minor component of the Plasmodium parasite, our findings demonstrate the important role of this phospholipid as a precursor of PE and PC, which are major constituents of parasite phospholipids.

Three clones have been prepared from the Honduras I/CDC strain of Plasmodium falciparum by a method of microscopic selection. One of these (HB-2) does not form gametocytes whereas the others (HB-1 and HB-3) do. All three are as resistant to pyrimethamine as the original line, and all three form knobs on the erythrocyte surface.

Malarial gametocytes, which are taken up by mosquitoes during a blood meal, develop in the gut of the mosquito into gametes. Gametes and gametocytes contain the target antigens of transmission-blocking immunity. Here, we show that the peripheral blood of nonexposed donors contains Plasmodium falciparum gamete-reactive T cells at frequencies ranging from 1/300 to 1/4000. Studies on long-term clones demonstrated that these cells often recognized antigens shared between gametes and asexual stage parasites or even between heterologous gametes, although it has been possible to derive a P. falciparum gamete-specific T clone. The T clones examined were T3+, T4+, T8-, and either HLA-DR- or HLA-DQ-restricted. They responded to gametes by both proliferation and the secretion of gamma-interferon. The gamete-specific clone and other asexual cross-reactive clones examined could be stimulated in vitro by a preparation of mature gametocytes within RBC, but not by RBC alone, suggesting that gametocytes are immunogenic or can become immunogenic for T cells in vivo. The significance of these observations to mosquito transmission of malaria and development and application of a gamete vaccine are discussed.

Asexual erythrocytic parasites of Plasmodium falciparum are killed by chloroquine, whilst mature gametocytes are not. The gametocytes of P. falciparum take 10 days to develop to maturity and their sensitivity to chloroquine during this time was studied in vitro to investigate when the switch from susceptibility to insusceptibility occurred and to compare the responses of asexual and immature sexual parasites to the drug. 45 to 50% of asexual parasites and immature gametocytes less than one day old survived in 0.1n. mols of chloroquine per ml but 0.3n. mols of drug per ml was lethal to both. Chloroquine at 1.0n mols per ml was lethal to developing gametocytes during their first six days of growth probably due, at least in part, to the drug disorganizing the parasite's digestion of host erythrocyte haemoglobin. The drug clumped the pigment of developing gametocytes. Only immature gametocytes in the final stage of development (stage 4) survive in high chloroquine concentrations.

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.

Sera collected from 176 children and adults from 3 different regions of Cameroon were used in inhibition assays against Plasmodium falciparum in vitro. The individuals had been classified clinically as malarious or non-malarious. Of the 58 non-malarious persons questioned, 39 claimed never to have had symptoms of the disease. Sera were tested for inhibitory effects on either merozoite entry and retardation of growth or inhibition of intraerythrocytic growth. The sera from malarious individuals showed only 9% and 29% mean inhibition values at 24 and 48 h, respectively, while those from the non-malarious persons showed 59% and 73% inhibition for the same periods. Of the 58 sera from non-malarious persons, 34 produced typical crisis forms by 24-36 h. Some sera also inhibited merozoite entry into erythrocytes. Our data suggest an important relationship between acquired immunity to P. falciparum in Cameroon and the types of inhibition described here.

The Haemosporidia of Mammals are classified in 3 groups according to the morphology of the gametocytes: 1) group "vivax" with Hepatocystis and several species of Plasmodium; 2) group "malariae" with Nycteria and a second group of Plasmodium; 3) group "falciparum" with only Plasmodium. In each group there is a correlation between the morphology of the gametocytes and that of the exo-erythrocytic schizonts. Through discussing the host-range and comparing the life cycles of Haemosporidia in Birds, Reptiles and Mammals, we suggest a hypothesis of the polyphyletism of Plasmodium of Mammals.

We report that synchronized cultures of Plasmodium falciparum with up to 40% parasitized cells can be obtained with the use of low, red blood cell suspensions and only daily replacement of culture medium. These cultures contained not only a reduced proportion of uninfected red cells but also a population of cells with brief and equal time of exposure to culture conditions. Such high parasitemias are desirable for studies of ring-staged parasites (for which enrichment techniques are not available) and late-staged parasites when the manipulations for enrichment are inappropriate or unsuccessful.

An in vitro model was developed to study the hepatic phase of Plasmodium falciparum, the only malaria parasite lethal to man. Primary cultures of human hepatocytes were inoculated with sporozoites of Brazilian and African strains of P. falciparum. On days 1 through 7 after inoculation examination of fluorescence-labeled and Giemsa-stained preparations demonstrated the presence of many intracellular parasites. In three separate sets of experiments all cultures were found to be infected with as many as 650 liver schizonts measuring up to 40 micrometers. After the addition of red blood cells, intraerythrocytic forms of P. falciparum were detected on days 12 and 13 by an immunofluorescence assay, indicating that the hepatic cycle had been completed in vitro.

Sera (BCG-lipopolysaccharide [LPS] serum) were obtained from mice infected with Mycobacterium bovis BCG 2 h after intravenous administration of bacterial endotoxin (LPS). Varying concentrations of sera were added to cultures of Plasmodium falciparum-infected human erythrocytes; parasite viability was assessed by hypoxanthine incorporation after 4 days in culture. At concentrations of 1 to 3%, cultures treated with BCG-LPS serum showed a two- to threefold increase in hypoxanthine incorporation; at higher concentrations (4 to 8%), hypoxanthine incorporation fell to 2 to 5% of that in control cultures. Concurrent assays with control sera (from untreated mice or mice treated with BCG or LPS alone) caused some stimulation but no inhibition at up to 8% concentration. Examination of cultures treated with BCG-LPS serum showed morphological, deterioration of parasites within erythrocytes. The presence of tumor necrosis factor in the BCG-LPS serum was confirmed by using a standard L-cell cytotoxicity assay. In addition, rabbit antiserum against partially purified tumor necrosis factor protected intraerythrocytic forms of P. falciparum from the toxic effects of BCG-LPS serum. These data suggest that the factor in BCG-LPS serum that is toxic to P. falciparum in human erythrocytes is antigenically similar or identical to tumor necrosis factor. This nonantibody mediator of killing may play a role in human malaria.

One-site and two-site immunoradiometric assays have been developed against an antigen on gametocytes of Plasmodium falciparum, using monoclonal antibodies (Mabs) which block transmission of the parasites to mosquitoes. Three such Mabs have been studied, each of which immunoprecipitates a complex of three gamete surface proteins of apparent Mr 260,000, 59,000, and 53,000 from Triton X-100 extracts of the parasites. The assays showed that the Mabs recognized one or the other of two distinct, nonrepeated epitopes on the target antigen(s). In the one-site assay certain combinations of two Mabs interacted at appropriate concentrations to enhance binding of the Mabs to the antigen. The same combinations of Mabs synergize to suppress infectivity of gametocytes to mosquitoes.

Resistance to mefloquine in Plasmodium falciparum has begun to occur along the border of Thailand and Kampuchea. As a means of assessing the natural occurrence of mefloquine resistance, the admission and post-treatment parasite isolates from a mefloquine treatment failure were cloned and characterized. Clones from the admission isolate were susceptible to mefloquine in vitro (ID50 of 3.4 [2-5], G [95% CI] ng/ml) and showed a mixture of isozyme types for glucose phosphate isomerase (GPI types I and II). The post-treatment clones were resistant to mefloquine in vitro (ID50 of 17.3 [13-23] ng/ml) with only one isozyme (GPI type I) detected. These observations suggest that under mefloquine pressure a resistant parasite population was selected in the patient, indicating that the potential for mefloquine resistance already exists in the indigenous P. falciparum gene pool. In addition, the mefloquine-resistant clones showed decreased susceptibility in vitro to halofantrine suggesting possible cross-resistance to this new antimalarial drug currently under development.