Antimalarial properties of bredinin. Prediction based on identification of differences in human host-parasite purine metabolism.

Human malaria parasites (Plasmodium falciparum) grown in continuous erythrocyte culture utilize hypoxanthine for synthesis of both guanosine and adenosine nucleotides. Unlike the mature human erythrocyte, the malaria parasite depends on a constant supply of guanylates, primarily for synthesis of nucleic acids. This parasite specific requirement for guanylates led us to predict that a block in the hypoxanthine to guanosine monophosphate pathway would be selectively lethal to the parasite. Bredinin (4-carbamoyl-1-beta-D-ribofuranyosyl-imidazolium-5-olate) inhibited the synthesis of guanosine monophosphate from inosine monophosphate by parasitized erythrocytes. This block in guanylate synthesis was fatal to both a drug-sensitive (FCR-3) and a drug-resistant (VNS) strain of the malaria parasite at a bredinin concentration of 50 microM, arresting growth of the parasite at the trophozoite stage of development. These studies emphasize the essential role of guanylates and their synthesis from hypoxanthine in the metabolism of malaria parasite. They further suggest that bredinin or similar agents that selectively interfere with parasite guanylate metabolism may have potential for antimalarial chemotherapy.

Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line.

In studies with the human promyelocytic leukemia cell line HL-60, we defined changes in intermediary purine metabolism that appear to contribute to the regulation of terminal maturation in myeloid cells. When HL-60 cells were exposed to compounds that induce maturation, consistent alterations in purine metabolism were found to occur within 24 h of culture. Perturbation of guanosine nucleotide synthesis and decreases of up to 50% in intracellular guanylate pool sizes were associated with the induced maturation of these cells in response to diverse inducing agents. While immature HL-60 cells were observed to synthesize purine nucleotides by both de novo and salvage pathways, the activity of both pathways decreased in cells induced to mature, although the relative contribution of purine salvage increased. Moreover, incorporation of the salvage pathway precursor, [14C]hypoxanthine from the intermediate, inosine monophosphate (IMP), into guanylates was reduced by approximately 65% in induced HL-60 cells, reflecting decreased activity of both hypoxanthine phosphoribosyltransferase and IMP dehydrogenase. When various inhibitors of IMP dehydrogenase (mycophenolic acid, 3-deazaguanosine, and 2-beta-D-ribofuranosylthiazole-4-carboxamide) were evaluated for their effects upon HL-60 cells, each agent was found to induce the cells to mature morphologically and functionally. Like other inducers, these agents decreased HL-60 cell proliferation and caused the cells to acquire an ability to phagocytose opsonized yeast and reduce nitroblue tetrazolium. Each agent reduced intracellular guanosine nucleotide pool sizes and induced HL-60 cell maturation at micromolar concentrations. These observations suggest that the size of intracellular guanosine nucleotide pools, the biosynthesis of guanosine nucleotides, and the activity of IMP dehydrogenase may be central to the regulation of terminal maturation in myeloid cells.

Pharmacokinetics of an extended-dose halofantrine regimen in patients with malaria and in healthy volunteers.

The pharmacokinetics and tolerance of a 4.5 gm 7-day halofantrine loading dose regimen were evaluated in 10 Thai patients with malaria and in 10 noninfected volunteers. Halofantrine peak plasma concentrations and bioavailability on the first day of treatment were significantly lower in patients with malaria than in healthy volunteers. Halofantrine elimination half-life was significantly shorter in patients with malaria than healthy control subjects (9.5 versus 15.8 days). These data show a distinct effect of acute malaria on the absorption and elimination of the drug. In addition, marked intersubject and intrasubject variability in peak and trough halofantrine levels was observed, indicating variable drug absorption. This dosing regimen was effective and well tolerated, with mild transient diarrhea during the first few days of treatment in both groups. To produce consistently effective drug levels, the currently recommended dosing regimens may be suboptimal. Slow halofantrine elimination raises concern for induction of parasite resistance when the drug is used in endemic areas of the world.

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum.

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.

Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species.

GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.

Polymorphism of proteins in malaria parasites following mefloquine treatment.

Proteins in malaria parasites (Plasmodium falciparum) isolated from a patient in Thailand before treatment, and after recrudescence of infection subsequent to mefloquine treatment, were compared by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis. Nine 'pre-treatment' and six 'recrudescent' clones were studied. Variants of the enzyme glucose phosphate isomerase were also noted and mefloquine susceptibility of each clone was measured by in vitro tests. The 'pre-treatment' isolate was found to contain at least four genetically distinct clones, all sensitive to mefloquine, while the 'recrudescent' isolate contained at least two other types of clone, both showing increased tolerance to mefloquine. These two more tolerant types of clone differed from all the sensitive ones studied in regard to several different protein variants as shown by 2D-PAGE analysis. It is concluded that at least two (and probably more) genetically distinct clones of parasites with increased tolerance to mefloquine were present in the parasite population before mefloquine treatment was given, and were selected under mefloquine pressure.

T cell responses in acute falciparum malaria.

T lymphocyte responses to malaria-specific antigens during acute falciparum malaria were studied to determine host-parasite interaction and its relation to the manifestations of the disease. The results indicate that while there is antigen-specific immunodepression, markedly elevated levels of soluble factors such as IL2 receptor, CD8 antigen and IFN-gamma suggest that there is intense concurrent cellular activation which however does not seem to be effective in controlling the infection. It is proposed that the cellular activation is to a large extent non-specific and polyclonal, and leads to the exaggerated production of cytokines and eventually immunopathology. Various mechanisms of immunodepression are discussed.

Increased gamma delta T cells in acute Plasmodium falciparum malaria.

The T cell receptor of gamma delta is normally expressed on a small percentage of peripheral lymphocytes. Although the role of gamma delta T cells in the physiologic immune response is still unknown, there is accumulating evidence that gamma delta T cells may participate in the immune response to mycobacterial and other infectious organisms. In this study, we have quantitated the number of circulating gamma delta T cells during acute Plasmodium falciparum malaria. The results indicate that gamma delta T cells are elevated during the acute infection and remain elevated for at least 4 weeks during convalescence. T cells may participate in the immune response against P. falciparum by functioning as non-MHC restricted cytotoxic cells against intraerythrocytic parasites. Alternatively, lymphokines may be produced on antigen stimulation which may have antiparasitic activity.

Plasmodium falciparum pigment induces monocytes to release high levels of tumor necrosis factor-alpha and interleukin-1 beta.

We show that high levels of tumor necrosis factor-alpha (TNF-alpha) activity were consistently detected when monocytes were cocultured with Plasmodium falciparum schizont stage-parasitized erythrocytes that subsequently ruptured. Isolated pigment recovered from ruptured schizonts was found to specifically induce monocyte release of high levels of TNF-alpha and interleukin-1 beta (IL-1 beta). Particulate free-culture supernatant that contained various soluble parasite macromolecules induced relatively low levels of TNF-alpha and IL-1 beta. When isolated pigment was treated with protease, the monokine inducing-activity was abolished. Isolated pigment prepared from different natural isolates of P. falciparum stimulated variable levels of monokine production. We propose that in vivo, malaria pigment from parasites sequestered in the host microvasculature is a physiologically relevant moiety that interacts with monocytes and stimulates the release of TNF-alpha and IL-1 beta. These observations suggest that malaria pigment may be a virulence factor in the monokine-mediated induction of organ-specific and systemic pathophysiology in falciparum malaria.

Naturally acquired circumsporozoite antibodies and their role in protection in endemic falciparum and vivax malaria.

The role of naturally acquired circumsporozoite (CS) antibodies in protection against falciparum and vivax malaria was evaluated in a group of Thai endemic villagers using a prospective cohort and a case-control study design. There was no evidence of protection by either the presence of positive CS antibody levels at the presumed time of sporozoite exposure or in individuals who persistently had measurable levels of the antibodies. The study defined levels of CS antibodies that were not protective in natural infection.

Rosette formation by Plasmodium coatneyi-infected red blood cells.

Animal models are needed for the study of cytoadherence in falciparum malaria. Red blood cell (RBC) rosette formation is one type of cytoadherence and appears to be associated with knob formation, endothelial cell adhesion and sequestration of Plasmodium-infected RBCs. Since Plasmodium coatneyi-infected RBCs develop knobs and sequester, we hypothesized that they also form rosettes. RBCs from P. coatneyi-infected rhesus monkeys (Macaca-mulatta) were collected, allowed to mature overnight in vitro and found to form rosettes as hypothesized. This observation adds to the known falciparum-like characteristics of P. coatneyi, and suggests that the Macaca mulatta-P. coatneyi model may be appropriate for pathophysiologic studies of cytoadherence.

Plasmodium coatneyi ring-infected erythrocyte surface antigens.

Plasmodium coatneyi produced ring-infected erythrocyte surface antigen (RESA) during infection of the rhesus monkey. This antigen was immunogenic and elicited an antibody response that was not persistent but was boosted by repeated infections in a manner similar to that seen in P. falciparum infections in humans. Preliminary data showed that the appearance and increasing titer of antibodies to P. coatneyi RESA-like antigen were associated with prolongation of intervals from inoculation to patency and with control of parasitemia. Studies using both immunofluorescence assay and Western blot analysis showed that P. coatneyi-immune rhesus serum cross-reacted with P. falciparum antigens, but P. falciparum immune human serum did not recognize P. coatneyi antigens in either assay. These results show that P. coatneyi expresses RESA-like antigen that elicits an antibody response similar to that observed for human antibody to P. falciparum RESA. However, antibodies to P. coatneyi did not cross-react with P. falciparum RESA in erythrocyte membrane immunofluorescence assay and dot immunoblot analysis, suggesting that different immunogenic epitopes are present on the two molecules. Our observations support the use of this primate model in RESA-based vaccine development.

Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand.

The therapy of Plasmodium falciparum malaria continues to be a problem in many parts of Southeast Asia because of multidrug resistance to nearly all existing antimalarial drugs. Atovaquone is a novel hydroxynaphthoquinone with broad spectrum anti-protozoal activity. We recently evaluated the antimalarial activity of atovaquone in a series of dose-ranging studies in 317 patients with malaria at the Bangkok Hospital for Tropical Diseases. Originally, the drug was administered alone. Using atovaquone alone resulted in satisfactory, initial clinical responses in all patients; the mean parasite and fever clearance times were 62 and 53 hr, respectively. However, irrespective of the duration of therapy, overall cure rates were approximately 67%. In vitro sensitivity studies on parasites taken from patients prior to treatment and at the time of recrudescence showed a marked decrease in susceptibility to atovaquone in the recrudescent parasites. To improve cure rates, atovaquone was administered in combination with other drugs with antimalarial activity. Proguanil and tetracycline were chosen due to laboratory evidence of potentiation; doxycycline was selected because it has a longer half-life than tetracycline. Although pyrimethamine did not show laboratory evidence of potentiation with atovaquone, it was chosen as an alternative inhibitor of dihydrofolic acid reductase with a longer half-life than proguanil. The clinical studies with these drug combinations confirmed the laboratory results with marked improvement in cure rates for proguanil, tetracycline, and doxycycline; pyrimethamine showed only minimal improvement. Proguanil was subsequently selected as the preferred drug partner because of its long record of safety and the ability to use the drug in pregnant women and children. Of the 104 patients with falciparum malaria treated with atovaquone plus proguanil for 3-7 days, 101 were cured and had virtually no adverse side effects. The combination of atovaquone and proguanil also was effective in eliminating erythrocytic forms of P. vivax, but parasitemia recurred in most patients.

Response of Plasmodium vivax variants to chloroquine as determined by microscopy and quantitative polymerase chain reaction.

Genotypic heterogeneity in the repetitive portion of the circumsporozoite (CS) protein of Plasmodium vivax has been reported from many P. vivax-endemic areas. The objective of this study was to determine if the VK210 and VK247 CS variants of P. vivax differed in their clearance rates following chloroquine (CQ) therapy. One hundred seventy-one cases of P. vivax infection occurring in patients presenting to a research treatment center in Thailand were analyzed. Finger-prick blood samples were collected for microscopy and spotted onto filter paper at presentation and on each of five days of observation through supervised CQ therapy. A portion of the CS gene was amplified from filter paper samples by the polymerase chain reaction (PCR) and genotyped by oligoprobes specific for the VK210 and VK247 CS repeat regions. The mean time to clear parasitemia as determined by thick blood smear was significantly longer for pure VK210 infections (51 hr; 95% confidence interval [CI] 47.4-54, P = 0.006) and mixed infections (53 hr; 95% CI 49.2-56.7, P = 0.0009) as compared with VK247 infections (44 hr; 95% CI 39.8-47.9). Five patients matched for parasitemia, age, sex, and previous malaria experience were selected from each of the three genotype groups in the larger study for further analysis by quantitative PCR of P. vivax genotype-specific DNA during a treatment course. The mean time to clear parasite DNA, as determined by PCR, was significantly slower for VK210 parasites (65 hr; 95% CI 51-79) than for VK247 parasites (47 hr; 95% CI 30-63, P = 0.045).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of naturally acquired antibody responses to a recombinant fragment from the N-terminus of Plasmodium falciparum glycoprotein 195.

Antibody responses to the glycoprotein precursor of the major merozoite surface antigens of Plasmodium falciparum (gp195) were investigated in acutely infected Thai adults. Specific IgG antibody was assayed by enzyme-linked immunosorbent assay using a recombinant fragment derived from the N-terminal region of gp195 as the capture antigen. Two control groups were found to be without significant cross-reacting antibody. Among occupationally exposed soldiers, 84 of 85 men developed positive antibody responses during acute falciparum malaria. Mean antibody levels began to increase at the time of diagnosis, peaking, often at high titers, within two weeks, and then decreased with an initial serum half-life of less than one month. The high frequency of gp195 antibody responses underscores a potential role in serodiagnosis, whereas the dynamic nature of the response suggests that a rigorous schedule of prospective serum sampling will be required to accurately assess the relationship between these antibodies and protection.

Characteristics of natural antibody responses to the circumsporozoite protein of Plasmodium vivax.

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with release of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.

Malaria chemoprophylaxis using proguanil/dapsone combinations on the Thai-Cambodian border.

The Thai-Cambodian border is a difficult area in which to provide adequate malaria chemoprophylaxis because of multiple drug-resistant Plasmodium falciparum. In 1990-1991, Thai soldiers were randomly selected to receive proguanil (200 mg/day) combined with dapsone (4 mg or 12.5 mg/day) (n = 184) or pyrimethamine/dapsone (12.5 mg and 100 mg/week) (n = 177). Doxycycline (100 mg/day) was given to men with glucose-6-phosphate dehydrogenase deficiency (n = 77). Falciparum malaria attack rates were the same whether proguanil/dapsone (10.3%) or pyrimethamine/dapsone (11.3%) was used. However, proguanil/dapsone was more effective than pyrimethamine/dapsone in preventing vivax malaria (1.6% versus 12.4%). Men receiving doxycycline had falciparum malaria (3.9%) and vivax malaria (1.3%) at low rates. Adjusting the dapsone component from 4 mg to 12.5 mg did not improve the prophylactic effectiveness. Hematologic toxicity was not observed with the proguanil/dapsone combination. We conclude that proguanil/dapsone is not a useful alternative for malaria chemoprophylaxis on the Thai-Cambodian border.

Emergence of multidrug-resistant Plasmodium falciparum in Thailand: in vitro tracking.

Mefloquine was introduced into Thailand in 1985 for the treatment of Plasmodium falciparum infection. Recently, clinical failure of mefloquine was observed in southeastern Thailand, where an epidemic of falciparum malaria occurred. Beginning in 1984 and continuing until 1989, in vitro monitoring of P. falciparum isolates from Borai, a border district in the southeastern part of the country, showed a progressive decrease in mefloquine sensitivity until 1989; in 1990, the degree and prevalence of resistance accelerated. A similar pattern of resistance was observed for halofantrine, an antimalarial drug not yet commercially available in Thailand. In vitro sensitivity patterns of mefloquine and halofantrine elsewhere in the country remained relatively unchanged. These observations suggest a serious deterioration in available drugs for the treatment of falciparum malaria in southeastern Thailand that is predicted to spread throughout the country and Southeast Asia.

Characterization of naturally acquired antibodies to the non-repetitive flanking regions of the circumsporozoite protein of Plasmodium falciparum.

Antibody responses to the circumsporozoite (CS) protein of Plasmodium falciparum have previously been reported against the central repeating tetrapeptides of this protein. Segments of the protein flanking the repeat region also contain B-cell epitopes, but specific antibody responses have not been previously characterized. Longitudinal serum sets from 16 Thai adults who developed acute falciparum malaria were selected to represent a spectrum of antibody response to the repeat region (R32). These sera were assayed by enzyme-linked immunosorbent assay using as capture antigen a recombinant fusion protein, NS1(81)RLF, which contains both flanking regions, but lacks the NANP and NVDP repeats of the P. falciparum CS protein. Antibody responses to the repeatless flanking (RLF) regions were observed in all subjects, including five individuals who lacked detectable anti-R32 antibody responses. Anti-RLF antibody responses induced by natural infection appear to be short-lived and of low-to-moderate magnitude. Thus, if anti-RLF antibodies prove to be protective, derived vaccine candidates may require presentation of these epitopes with adjuvants or delivery systems that enhance immunogenicity.

Circumsporozoite antibody as a serologic marker of Plasmodium falciparum transmission.

In a longitudinal study of a malaria-endemic village in southeastern Thailand, circumsporozoite (CS) antibody to sporozoites of Plasmodium falciparum was measured by an enzyme-linked immunosorbent assay to determine its usefulness as a seroepidemiologic marker of malaria transmission. The CS anti-(NANP)n antibody level and prevalence during a 25-month period paralleled the pattern of seasonal transmission consistent with conventional parasitologic and entomologic measurements. The prevalence and level of antibody decreased during the non-transmission wet season, and increased over a 1-2-month transition period between the end of monsoon rains and the onset of dry conditions, an interval of maximum vector activity. Antibody increased with age in the population. The prevalence of antibody to the asexual blood stage as measured by conventional indirect fluorescent antibody assay did not coincide with changes in transmission and was sustained throughout the study period. Thus, CS antibody appeared to reflect the relative population exposure to mosquito inoculation of P. falciparum sporozoites and provided a useful measure of malaria transmission dynamics.