Qinghaosu-induced changes in the morphology of Plasmodium inui

The ultrastructural changes induced by the administration of the antimalarial drug, qinghaosu, were studied in monkeys (Macaca assamensis) infected with Plasmodium inui. Significant changes, notably mitochondrial swelling within the parasites but not within host cells, were first observed 2.5 hr after exposure to qinghaosu. This suggests that the target of qinghaosu may be the parasite's mitochondria, as occurs with primaquine. This is in contrast to the most widely used antimalarial drug, chloroquine.     

CHLOROQUINE RESISTANCE IN MALARIA: A DEFICIENCY OF CHLOROQUINE BINDING

Chloroquine-(14)C was used to study the processes which concentrate chloroquine in mouse red blood cells infected with chloroquine-sensitive or with chloroquine-resistant Plasmodium berghei. The initial rates of uptake and exchange of chloroquine-(14)C were both too fast to measure, yet large concentration gradients were maintained by the cells. When red blood cells were exposed to 10(-8)M chloroquine at 22 degrees C, with pH between 7.2 and 7.4, steady-state gradients of chloroquine-(14)C were approximately 600:1 (cells:medium) for cells infected with chloroquine-sensitive parasites, 100:1 for cells comparably infected with chloroquine-resistant parasites, and 14:1 for uninfected cells. The processes responsible for these gradients were saturable, in agreement with the proposal of chloroquine binding to cellular constituents. No degradation of chloroquine was detected.The major difference between the chloroquine-sensitive and -resistant parasites was deficiency of high-affinity binding of chloroquine by cells infected with chloroquine-resistant parasites. This deficiency explains the reduced ability of chloroquine-resistant parasites to concentrate chloroquine, and it suggests that chloroquine resistance is due to a decrease in the number, affinity, or accessibility of chloroquine receptor sites on a constituent of the malaria parasite.     

Preliminary report on the use of desferrioxamine in the treatment of Plasmodium falciparum malaria

The administration of 0.5 g (IM) of desferrioxamine every 12 hr for 3 days with 25 mg/kg (total dose for three days) of chloroquine abated the parasitemia of Plasmodium falciparum infected individuals more rapidly than chloroquine alone. Two patients with in vitro evidence of total or partial resistance to chloroquine also were successfully treated with the combination of drugs, and were free of parasitized red cells by day 7. Further clinical trials and development of desferrioxamine in the treatment of P. falciparum are warranted.     

Infection of Anopheles darlingi fed on patients infected with Plasmodium vivax before and during treatment with chloroquine in Costa Marques, Rondonia, Brazil

Patients with asexual and sexual parasites of Plasmodium vivax were treated orally with 600 mg of chloroquine diphosphate at hour zero, followed by 300 mg at 8, 24 and 48 hr. Anopheles darlingi were fed before the first dose (-0.5 hr) and at 0.5, 1, 2, 4, 6, 8, 9, 10, 11, 12, 20, 24, 36, 48, 60, and 72 hr later. Mosquitoes were examined for oocysts on day 8 and sporozoites on day 15 after infection. The frequency of infected mosquitoes and the mean number of oocysts were lower in mosquitoes that fed on patients 2-4 hr after the initial dose of chloroquine than in mosquitoes fed before treatment and at 0.5 and 1 hr. This sporontocidal effect was temporary, since the frequency of infected mosquitoes and the mean number of oocysts increased in mosquitoes fed 4-8 hr after the first dose. Nearly all mosquitoes fed on patients after the third dose of chloroquine, at 24 hr, were negative for P. vivax oocysts. Oocysts in mosquitoes fed on patients before, during, and after chloroquine treatment appeared normal and produced sporozoites. We conclude that chloroquine affects either the gametocytes, fertilization, zygotes, and/or ookinetes of P. vivax, but not subsequent stages of development.     

Potent antimalarial activity of clotrimazole in in vitro cultures of Plasmodium falciparum

The increasing resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands a continuous effort to develop new antimalarial agents. In this quest, the identification of antimalarial effects of drugs already in use for other therapies represents an attractive approach with potentially rapid clinical application. We have found that the extensively used antimycotic drug clotrimazole (CLT) effectively and rapidly inhibited parasite growth in five different strains of P. falciparum, in vitro, irrespective of their chloroquine sensitivity. The concentrations for 50% inhibition (IC(50)), assessed by parasite incorporation of [(3)H]hypoxanthine, were between 0.2 and 1.1 microM. CLT concentrations of 2 microM and above caused a sharp decline in parasitemia, complete inhibition of parasite replication, and destruction of parasites and host cells within a single intraerythrocytic asexual cycle (approximately 48 hr). These concentrations are within the plasma levels known to be attained in humans after oral administration of the drug. The effects were associated with distinct morphological changes. Transient exposure of ring-stage parasites to 2.5 microM CLT for a period of 12 hr caused a delay in development in a fraction of parasites that reverted to normal after drug removal; 24-hr exposure to the same concentration caused total destruction of parasites and parasitized cells. Chloroquine antagonized the effects of CLT whereas mefloquine was synergistic. The present study suggests that CLT holds much promise as an antimalarial agent and that it is suitable for a clinical study in P. falciparum malaria.     

A rapid in vitro test for chloroquine-resistant Plasmodium falciparum

We report a rapid (2-3 hr) in vitro test for chloroquine resistance in Plasmodium falciparum. The test is based on the inhibition of chloroquine efflux by verapamil; it is performed by diluting infected blood in culture medium and incubating the diluted blood for 60 min at 37 degrees C with 50 nM 3H-chloroquine, with and without 10 microM verapamil. The test can be performed with the ring stage parasites in the blood of infected patients and in the presence of white cells, platelets and anticoagulants (heparin, EDTA, or citrate). Although the test can be performed in triplicate with 20 microliters of blood and specimens may be kept in anticoagulants at 4 degrees C for up to 24 hr, parasitemias less than 0.1% limit the sensitivity of the assay. Inhibition of chloroquine efflux by verapamil may permit the rapid identification of chloroquine resistant P. falciparum in blood specimens from infected patients.     

In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.     

Chloroquine concentration profile in the community of Mewat region, district Gurgaon (Haryana), India

A survey was conducted to find chloroquine concentration profile in the community of Mewat region district Gurgaon (Haryana) of India. 88 P. falciparum and 3 P. vivax cases were detected out of 148 blood slides examined with a SPR of 61.48. Plasma chloroquine and desethylchloroquine concentrations were determined in 55 P. falciparum and 2 P. vivax patients and 29 persons whose blood slides were negative for malaria parasite before giving any treatment. Mean chloroquine concentrations in cases with P. falciparum parasites and without malaria parasites were 0.018 and 0.016 microg ml(-1) respectively. Chloroquine to desethyl chloroquine ratio was between 2 and 3 in both groups. Only 10 malaria parasite negative cases out of 29 had plasma chloroquine concentrations above 0.016 microg ml(-1) required for malaria chemoprophylaxis. Chloroquine was undetectable in plasma samples of 8 out of 55 P. falciparum cases. Chloroquine plasma concentrations in 21 P. falciparum cases were below therapeutically effective concentration of 0.016 microg ml(-1) suggesting improper treatment while in 29 P. falciparum cases, parasitemia recurred despite required chloroquine concentration confirming chloroquine resistant status. Irregular prophylaxis and lack of proper treatment was one of the major causes of malaria outbreak in this area.     

The chemical composition of normal human red blood cells,including variability among centrifuged cells

1. Red cells from different layers of centrifuged cells vary in composition.Cells obtained from the upper layer, which is relatively richer in reticulocytes,contain more water, sodium, potassium, chloride and phosphorus than the remaining cells.

2. The direct method of analysis of red blood cells using a constricted typecentrifuge tube to separate the entire red cells sample from buffy layer cells andfrom plasma avoids the errors in direct analysis caused by different cell population in upper and lower layers of centrifuged cells and the cumulative errorsinherent in indirect analysis.

3. Using the direct method and a constricted type centrifuge tube, the meansand standard deviations of the water and mineral content of the erythrocytesand plasma of 11 normal males and 11 normal females were determined. Maleswere found to have a higher sodium content of red cells and plasma.

4. The sum of the molal concentrations of sodium, potassium, chloride andphosphorus in red cells is not always equal to the sum of the molal concentrations of these minerals in the plasma.

Submitted on April 20, 1954 Accepted on June 11, 1954     

Excess hemoglobin digestion and the osmotic stability of Plasmodium falciparum-infected red blood cells

During their asexual reproduction cycle (about 48 hours) in human red cells, Plasmodium falciparum parasites consume most of the host cell hemoglobin, far more than they require for protein biosynthesis. They also induce a large increase in the permeability of the host cell plasma membrane to allow for an increased traffic of nutrients and waste products. Why do the parasites digest hemoglobin in such excess? And how can infected red cells retain their integrity for the duration of the asexual cycle when comparably permeabilized uninfected cells hemolyse earlier? To address these questions we encoded the multiplicity of factors known to influence host cell volume in a mathematical model of the homeostasis of a parasitized red cell. The predicted volume changes were subjected to thorough experimental tests by monitoring the stage-related changes in the osmotic fragility of infected red cell populations. The results supported the model predictions of biphasic volume changes comprising transient shrinkage of infected cells with young trophozoites followed by continuous volume increase to about 10% lower than the critical hemolytic volume of approximately 150 fL by the end of the asexual cycle. Analysis of these results and of additional model predictions demonstrated that the osmotic stability of infected red cells can be preserved only by a large reduction in impermeant solute concentration within the host cell compartment. Thus, excess hemoglobin consumption represents an essential evolutionary strategy to prevent the premature hemolysis of the highly permeabilized infected red cell.     

咯萘啶,阿莫地喹,甲氟喹及青蒿素对伯氏疟原虫..

The asexual stages of P. berghei ANKA were completely eliminated as revealed in a "4-day suppressive test" with the daily dose of pyronaridine 12.5 mg base/kg or amodiaquine 25 mg base/kg. Mefloquine 25 mg base/kg and qinghaosu 100 mg/kg though exerted obvious suppressive effect, the cure rates were only 50% and 0%, respectively. In treating chloroquine-sensitive P. berghei ANKA strain pyronaridine exhibited the best therapeutic activity, which was followed by amodiaquine, mefloquine and quinghaosu. In treating moderately chloroquine-resistant P. berghei NS line the cure rate of pyronaridine 12.5 mg/kg.d x 4 was 70%, but none of the 10 infected mice from any group was cured by amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d or qinghaosu 200 mg/kg.d. Though the latter 3 drugs showed prominent suppressive effects, parasitemia remained positive or recrudesced after dosing. We demonstrate that parasites resistant to chloroquine had cross resistance to amodiaquine, mefloquine and inghaosu at various degrees. Amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d and qinghaosu 200 mg/kg.d exhibited no obvious suppressive activity on highly pyronaridine-resistant line of P. berghei, indicating the existence of cross resistance to pyronaridine.     

Altered plasma membrane phospholipid organization in Plasmodium falciparum-infected human erythrocytes

The intraerythrocytic development of the malaria parasite is accompanied by distinct morphological and biochemical changes in the host cell membrane, yet little is known about development-related alterations in the transbilayer organization of membrane phospholipids in parasitized cells. This question was examined in human red cells infected with Plasmodium falciparum. Normal red cells were infected with strain FCR3 or with clonal derivatives that either produce (K+) or do not produce (K-) knobby protuberances on the infected red cells. Parasitized cells were harvested at various stages of parasite development, and the bilayer orientation of red cell membrane phospholipids was determined chemically using 2,4,6-trinitrobenzene sulphonic acid (TNBS) or enzymatically using bee venom phospholipase A2 (PLA2) and sphingomyelinase C (SMC). We found that parasite development was accompanied by distinct alterations in the red cell membrane transbilayer distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). Increases in the exoplasmic membrane leaflet exposure of PE and PS were larger in the late-stage parasitized cells than in the early-stage parasitized cells. Similar results were obtained for PE membrane distribution using either chemical (TNBS) or enzymatic (PLA2 plus SMC) methods, although changes in PS distribution were observed only with TNBS. Uninfected cohort cells derived from mixed populations of infected and uninfected cells exhibited normal patterns of membrane phospholipid organization. The observed alterations in P falciparum-infected red cell membrane phospholipid distribution, which is independent of the presence or absence of knobby protuberances, might be associated with the drastic changes in cell membrane permeability and susceptibility to early hemolysis observed in the late stages of parasite development.     

The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites

Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6- phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.     

弓形虫RH株在实验感染小鼠体内分布的研究

以间接免疫酶法观察弓形虫感染时的急性期病变特点及虫体在主体脏器的动态分布,以期为弓形虫病的病理诊断提供依据,并对弓形虫致病机理加深理解。方法用弓形虫ＲＨ株速殖子１０＾３个经腹腔感染昆明小鼠,于感染后第２、４、６和８ｄ,取肝、脾、肺和脑进行间接免疫酶染色。     

Electron probe analysis of sodium and other elements in hypertrophied and sodium-loaded smooth muscle

The composition of normal, hypertrophied, or sodium-loaded rabbit portal anterior mesenteric vein and of normal and sodium-loaded guinea pig taenia coli smooth muscle was measured in cryosections with electron probe analysis, and the effects of wash with cold sodium-free (Lithium) solutions were determined. There was no significant difference in the cytoplasmic, nuclear, or mitochondrial concentrations of any of the measured elements (sodium, potassium, chloride, magnesium, calcium, phosphorus, sulfur) between hypertrophied, sham-operated, or control veins. The cytoplasmic potassium:sodium:chloride ratio in rabbit portal anterior mesenteric vein was 1:0.26:0.46, and the average sodium concentration (198 mmol/kg dry cytoplasmic weight) was nearly twice as high as estimated from ion flux measurements. The cytoplasmic sodium concentration of normal guinea pig taenia coli was 61 mmol/kg dry weight. The existence of a rapidly exchanging, relatively low affinity, and temperature-insensitive component of cytoplasmic sodium efflux was indicated by the reduction in cytoplasmic sodium after washout in cold, sodium-free (lithium or Tris-substituted) solutions. This reduction, by 62% in normal, 71% in sodium-loaded portal anterior mesenteric vein, and 36% in sodium-loaded guinea pig taenia coli smooth muscle, suggests that the lithium wash method can underestimate cell sodium. In sodium-loaded guinea pig taenia coli and portal anterior mesenteric vein smooth muscles, the cytoplasmic sodium analyzed in individual cells showed a bimodal distribution; in cryosections, the cells having the highest sodium and lowest potassium and phosphorus content also had a more electronlucent (light cell) appearance.     

Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell.

Erythroid cells which are homogeneous with regard to stage of maturation are naturally available from the circulation of chick embryos at various times of development. This provides a convenient system for examining the changes in plasma membrane protein composition during red cell maturation. Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive characterization of the isolated membranes show that they are pure and their proteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both qualitative and quantitative changes occur in membrane protein composition during the early stage of erythroid differentiation. Specific proteins of red cell membrane such as "spectrin" and band three proteins are present in low levels in early erythroblasts but increase in their relative amounts with maturation. A steady-state membrane protein composition seems to be established by the late polychromatophilic erythroblast stage.     

Cation Content and Membrane Deformability of Heterozygous P-Thalassaemic Red Blood Cells

S ummary . The red blood cells of patients heterozygous for 8-thalassaemia contain less sodium, potassium and water than do normal red cells. Incubation of red cells in plasma at 37°C for 24 hr in the absence of glucose produced the following changes: (1) An increase in the red-cell sodium content of both normal and thalas-saemic cells. (2) A decrease in the red cell potassium content which was much more pronounced in thalassaemic than in normal cells. (3) A reduction in the total cation content of the red cells from patients heterozygous for β-thalassaemia; this reduction seemed to be related to the clinical severity of the disease, as well as the reduction in mean corpuscular volume and osmotic fragility. (4) The trapped plasma values, which reflect the deformability of red cells, were initially greater for thalassaemic red cells than normal cells and in both groups increased after incubation.
The addition of glucose to the incubation medium largely prevented the changes produced by incubation. The changes induced by incubation in vitro may be relevant to changes in vivo , where a local lack of glucose might lead to a loss of potassium and water from thalassaemic cells. This could give rise to shrinking of the red cells and a reduction in their deformability which may play a role in the haemolysis of heterozygous β-thalassaemia.     

Eosinophilia, anemia and parasitism in a rural region of northwest Thailand.

Parasitic infections are prevalent in Thailand. The parasites' relationship with anemia and eosinophilia has been rarely reported due to limited health care access, especially in rural areas. We studied the prevalence and relationship between eosinophilia, anemia and parasitism in 169 Thai-Karens from Mae Lamung and Mae Chan subdistricts, Umphang district, Tak Province, using an automated complete blood counter, and microscopic examination for intestinal parasites and microfilaria. In Mae Chan, 5 individuals were microfilaremic, 72% of individuals examined were infected with at least one kind of intestinal parasites, and 50% were anemic, with normal mean red cell volume (MCV). In Mae Lamung, 46% were parasitized (Triteeraprapab et al, 1997) but none were microfilaremic or anemic. In both populations, eosinophilia was prevalent (77%). Hookworm infection was found to be significantly associated with eosinophilia, but not anemia nor microcytosis of red cells.     

Investigation of the in vitro Gender-Specific Partitioning of Mefloquine in Malarial Infected Red Blood Cells and Plasma

The investigation of gender-specific partitioning of the antimalarial drug mefloquine to cellular and fluid blood compartments was performed using blood collected from a female and male healthy subject that were infected with Plasmodium falciparum PCM2 clone and spiked with mefloquine (0.25, 1, and 5 μM). Mefloquine concentrations in red cells of both female and male subjects were significantly higher than plasma, which suggests an intensive uptake by red cells. This was supported by a high ratio of mefloquine concentrations in the parasitized and non-parasitized red cells of about 4-fold. Gender-specific partitioning of mefloquine in parasitized blood was seen only in plasma where significantly higher concentrations were observed in female compared with male plasma. Down-adjusting the therapeutic dose of mefloquine in female patients with malaria is not advisable because mefloquine concentrations in the target cellular compartment are similar in both genders.