Brain endothelial cells increase the proliferation of Plasmodium falciparum through production of soluble factors.

We here describe the novel finding that brain endothelial cells in vitro can stimulate the growth of Plasmodium falciparum through the production of low molecular weight growth factors. By using a conditioned medium approach, we show that the brain endothelial cells continued to release these factors over time. If this mirrors the in vivo situation, these growth factors potentially would provide an advantage, in terms of enhanced growth, for sequestered parasitised red blood cells in the brain microvasculature. We observed this phenomenon with brain endothelial cells from several sources as well as a second P. falciparum strain. The characteristics of the growth factors included: <3 kDa molecular weight, heat stable, and in part chloroform soluble. Future efforts should be directed at identifying these growth factors, since blocking their production or actions might be of benefit for reducing parasite load and, hence, malaria pathology.

Conventional and experimental treatment of cerebral malaria.

The most severe complication of Plasmodium falciparum infection is cerebral malaria (CM). Cerebral malaria implies the presence of neurological features, especially impaired consciousness. The treatment of CM is limited to: (i) a few conventional anti-malarial drugs (quinine or artemisinins), (ii) adjunctive treatments (initial stabilisation, blood exchange transfusion, osmotic diuretics and correction of hypoglycaemia, acidosis and hypovolaemia) and (iii) immunomodulation. There are clear procedures concerning treatment of CM, which include the use of the anti-plasmodial drugs. Adjunctive treatments are permissible but there is no single official guideline and immune intervention is a possibility currently being examined in rodent models only. The suggested immunomodulation approach is based on the strong likelihood that CM is the result of an immunopathological process. P. falciparum initiates the multifactorial chain of events leading to lethal CM and, after a certain stage, it is impossible to stop the progression even by using anti-malarial drugs. We present evidence that CM is a result of a dysregulated immune response. Therefore, it might be prevented by early modulation of discrete factors that participate in this process. In experimental systems, some immunomodulators delay or prevent CM without affecting the parasitaemia. Therefore, in the future the ultimate treatment of CM may be a combination of an anti-malarial and an immunomodulator. However, the overall effect of an immunomodulator would need to be carefully examined in view of concomitant infections, especially in malaria endemic areas.

New formulations and derivatives of amphotericin B for treatment of leishmaniasis.

The clinical treatment of leishmaniasis is based on a limited number of drugs, which are associated with adverse effects and have already induced resistance. Amphotericin B (AmB), a polyene antibiotic produced by Streptomyces sp, is the only anti-leishmanial drug which has not induced clinical resistance since its discovery in 1956. The limiting factor in the use of AmB is its toxic effects, mainly nephrotoxicity. The maximal dose of AmB for human use is 1.5 mg/kg which sometimes is not sufficient for cure. The mode of action of AmB is associated with its toxicity: it selectively binds to parasite membrane ergosterol but also, to a lesser extent, to human cholesterol. Apart from this mechanism, AmB has immunomodulatory effects, some of them are deleterious. Reduction of the toxic effects by using lipid formulations allows the infusion of higher doses of AmB. Unfortunately, these formulations are relatively expensive and therefore out of reach for patients in need, in the endemic areas. All the existing formulations are given parenterally, which has obvious disadvantages; most important is the need for hospitalization or multiple visits in the clinic. The current efforts to improve AmB are directed at the production of AmB aggregates in liquid solutions, encapsulation with lipid components, and solubilization by binding to soluble polymers. The expected improved treatment resulting from use of the new formulations is based on better pharmacokinetics, reduced toxicity originating from slow release, targeting to the infected organ and an altered pattern of immune responses (related to AmB). Of particular importance are the attempts to produce derivatives for oral treatment, which will decrease costs of hospitalization and improve applicability for children and the elderly population.

The effect of a live Neospora caninum tachyzoite vaccine in naturally infected pregnant dairy cows.

Neosporosis, caused by the intracellular protozoan Neospora caninum, is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum-seropositive pregnant dams were inoculated with 10(8) live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively (P=0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows.

Transgenic mice with elevated level of CuZnSOD are highly susceptible to malaria infection.

Copper/zinc superoxide dismutase (CuZnSOD) catalyses the conversion of O2.- into H2O2. Constitutive overexpression of CuZnSOD in cells and animals creates an indigenous oxidative stress that predisposes them to added insults. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of CuZnSOD affected the susceptibility of these mice to plasmodium infection. Acute malaria is associated with oxidative stress, mediated by redox-active iron released from the infected RBC. Two independently derived Tg-CuZnSOD lines showed higher sensitivity than control mice to infection by Plasmodium berghei (P. berghei), reflected by an earlier onset and increased rate of mortality. Nevertheless, while Tg-CuZnSOD mice were more vulnerable than control mice, the levels of parasitemia were comparable in both strains. Moreover, treatment of infected red blood cells (RBC) with oxidative stress inducers, such as ascorbate or paraquat, reduced the viability of parasites equally in both transgenic and control RBC. This further confirms that increased CuZnSOD does not support plasmodia development. The data are consistent with the possibility that the combination of increased redox-active iron and elevated H2O2 in the plasmodium-infected Tg-CuZnSOD mice, led to an enhanced Fenton's reaction-mediated HO. production, and the resulting oxidative injury renders the transgenic mice more vulnerable to parasite infection.

Detection of Plasmodium falciparum using a radioimmunoassay based on a crossreacting, monoclonal anti-P. berghei antibody-P. berghei antigen system.

An antibody binding-inhibition test is described, which allows the detection of P. falciparum in red blood cells (RBC) infected in vitro, using a crossreacting, monoclonal anti-P. berghei antibody and P. berghei coated microtiter plates. Experiments carried out to determine the coating efficiency of various P. berghei and P. falciparum derived antigen preparations showed that intact, saponin freed P. berghei parasites and sonicated, RBC parasitized with P. falciparum had the highest binding activity. Binding of the monoclonal antibody to the antigen coated plates was effectively inhibited by preincubation with sonicated, P. falciparum infected RBC. The minimal degree of infection detectable was about 0.008% parasitemia (400 parasitized RBC/microliters blood). The sensitivity of detection was not appreciably affected by the source of the coating antigen. We conclude that the difficulty and expense involved in the use of P. falciparum based immunodiagnostic tests for large scale screening for malaria can be obviated by making use of P. berghei based assays.

A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies.

A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10(6) RBC.

Use of a monoclonal, anti Plasmodium berghei antibody, cross reacting with P. falciparum, for the detection of P. falciparum in in vitro infected blood.

This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.

Optimisation of flow cytometric measurement of parasitaemia in plasmodium-infected mice.

Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei.

Deleterious synergistic effects of ascorbate and copper on the development of Plasmodium falciparum: an in vitro study in normal and in G6PD-deficient erythrocytes.

The effects of ascorbate and copper on the development of Plasmodium falciparum were studied in two modes: pretreatment of uninfected erythrocytes followed by infection by P. falciparum and treatment of parasitized erythrocytes. Pretreatment of G6PD(+) cells with ascorbate caused a slight enhancement in parasite development, while in G6PD(-) cells a suppressive effect on the plasmodia was demonstrated. Copper alone interfered with parasite growth in both cell types. The combination of copper and ascorbate arrested parasite maturation, an effect which was more pronounced in G6PD(-) cells. Synergism between copper and ascorbate was better demonstrated following the treatment of infected erythrocytes: while ascorbate alone supported parasite development and copper alone had only a marginal suppressive effect, the combination of copper and ascorbate yielded a marked inhibition of parasite growth. Ascorbate proved destructive to the parasites in the presence of adventitious copper, or on the second day of the parasite life cycle. In these cases it acted as a pro-oxidant, while in other systems, in particular in the presence of a chelator, ascorbate acted as an antioxidant and promoted parasite growth. The understanding of the role of transition metals and free radicals in parasite development and injury could shed light on novel approaches to fight malaria.

Synthesis and biodegradation of arabinogalactan sponges prepared by reductive amination.

The synthesis of polysaccharide-based sponges for the use in tissue engineering was systematically investigated. A comparison study of the branched polysaccharide arabinogalactan (AG) and the linear polysaccharide dextran in the formation of sponges by the reaction with diamines or polyamines was conducted. Three AG-based sponges were synthesized from the crosslinking reaction with different amine molecules. The sponges obtained were highly porous, rapidly swelled in water, and were stable in vitro for at least 11 weeks in aqueous media at 37 degrees C. AG-chitosan sponges were chosen as most suitable to serve as scaffolds for cell growth in tissue engineering. The biocompatibility in vivo of these sponges was evaluated by histological staining and non-invasive MRI technique after implantation in BALB/c mice. The sponge evoked an inflammatory response with vascularization of the implant. The inflammatory reaction decreased with time, indicating a healing process.

Synthesis and characterization of novel water soluble amphotericin B-arabinogalactan conjugates.

The coupling of amphotericin B (AmB), a water-insoluble antifungal agent, to arabinogalactan (AG) via an imine or amine bond was systematically investigated. AG was oxidized using potassium periodate, purified from the oxidizing agent using ion-exchange chromatography, and reacted with AmB to form the Schiff base. The Schiff base was reduced to the amine using borohydride. All reactions took place in aqueous media. The purification of the oxidized AG from the oxidizing agent was essential to prevent oxidative degradation of AmB at the coupling step. We investigated the effects of AmB to AG ratio, buffer type, and reaction pH on the reaction yield, molecular weight, conjugate activity against pathogenic yeast and hemolytic activity. The optimum coupling conditions were buffer borate 0.1 M, pH 11 at room temperature for 48 h. Lower toxicity in vivo was achieved by using low-pressure gel permeation chromatography and applying the solution of AmB-AG conjugate through a Sephadex column. Both amine and imine AmB-AG conjugates were soluble in water and exhibited improved stability in aqueous solutions as compared to the unbound drug. The conjugates showed comparable minimum inhibitory concentration (MIC) values against Candida albicans. The conjugates were about 60 times less hemolytic against sheep erythrocytes than the free drug, and about 40 times less toxic in BALB/c mice.

Typing of southern African isolates of Plasmodium falciparum using monoclonal antibodies.

Antigenic diversity among 19 southern African isolates of Plasmodium falciparum was demonstrated using a panel of 9 monoclonal antibodies. Parasites obtained from single patients were heterogeneous. The antigen composition of 9 isolates was not stable with time in culture, particularly not with respect to 4 of the monoclonal antibodies. By the end of the investigation, 70% of isolates displayed an identical antigen pattern which was markedly different to any obtained in other parts of the world. Differences may be due to geographic origin of parasites or to variation in culture conditions.

A radioimmunoassay for the diagnosis of malaria.

A newly developed radioimmunoassay for the diagnosis of malaria has been tested in South Africa. The radioimmunoassay is an antibody binding-inhibition assay, based on a monoclonal antibody (D5) cross-reacting with Plasmodium berghei and P. residual binding activity was tested on antigen-coated microtiter plates. A sample was considered positive if it inhibited binding of the antibodies to an extent exceeding that of the microscopically negative blood samples. Blood was collected on 3 separate occasions from a total of 530 individuals living in a malaria-endemic area and was examined by radioimmunoassay and microscopy. Group 1, consisting of 194 samples, yielded 12 samples positive by microscopy and 10 of these (83%) were also positive by radioimmunoassay. One sample in this group was "positive" in the radioimmunoassay but negative on microscopy (false positive). In the 320 samples of group 2, 13 were positive by microscopy and 6 (46%) by radioimmunoassay. Group 3, which included 16 samples preselected as positive by microscopic examination and 16 controls, was examined after 4 weeks storage at -20 degrees C. Twelve samples (75%) were positive by radioimmunoassay. Tests carried out to determine the effect of blood storage on the activity of the antigen indicated that activity was preserved with little loss over a 3-month period.

Detection of Plasmodium falciparum in blood using DNA hybridization.

A rapid and simple assay for detecting Plasmodium falciparum in human blood was developed. The assay is based on DNA-DNA spot hybridization, using radiolabeled P. falciparum DNA as a probe and finger prick blood as the assay sample. It is very sensitive, able to detect parasitemia levels of 0.0001% in 10 microliter of blood. The assay can be quantified and used to estimate parasitemia levels. Several hundred blood samples can be processed simultaneously, and the entire procedure is completed within 24 hr. This assay can be useful for epidemiological surveys, for screening of blood by blood banks and for health authorities examining immigrants and tourists coming from malaria infested areas.

Preliminary field trial of a radioimmunoassay for the diagnosis of malaria.

A radioimmunoassay (RIA) has been developed for the detection of Plasmodium falciparum in infected blood. The assay is based on the ability of solubilized, infected red blood cells (RBC) (P. falciparum "antigen") to combine with anti-P. falciparum antibodies and thus prevent the subsequent interaction of the latter with "antigen"-coated microtiter plates. A preliminary trial was carried out in Thailand to determine the usefulness of the RIA for the immunodiagnosis of malaria. Blood samples from malarious and non-malarious patients were examined both by standard microscopy and by RIA. Efficient solubilization of the parasites proved to be a major requirement for the successful performance of the RIA. Sonication or freezing and thawing, which were perfectly satisfactory for the solubilization of cultured, infected RBC, were found to be totally inadequate when applied to RBC taken from patients. However, parasites in RBC from patients could be solubilized efficiently by treatment with detergents (e.g., NP40, Triton X-100, etc.). Of the 108 blood samples tested, 23 were found positive for falciparum parasitemia by microscopy and 39 by RIA. One sample from a patient with patent falciparum parasitemia and three with patent vivax parasitemia were negative by RIA. Ten of the samples positive only by RIA belonged to patients with recent malarial infection, as shown by microscopy. Thus, the RIA detected almost all of the patients with microscopic evidence of falciparum malaria. The proportion of false positives in the RIA test was low.

Neutralizing antibody in Rodent Malaria.

Small numbers of rat erythrocytes infected with viable P. berghei, when inoculated into susceptible rats together with hyperimmune rat serum (HIS), are fully neutralized. Serum from convalescent rats delays the onset of patency but does not neutralize. The neutralizing efficiency of HIS rises in proportion to the number of successive reinoculations of hyperimmune rats. In contrast, mice inoculated with parasites together with either HIS or normal rat serum succumbed to the disease at the same time after inoculation. Neutralization in rats occurs in vivo and is completed within 22 hours of inoculation. Much larger amounts of HIS are needed to achieve neutralization in splenectomized recipients than in intact rats. The action of HIS is dose-dependent. Thus, the degree of suppression of parasitaemia is proportional to the dose of HIS, while the mortality rate is inversely proportional to the dose. Suboptimal doses may even enhance the infection of recipient rats. The ability to produce neutralizing antibody is dissimilar in two strains of rat. Thus, the outbred Sabra strain produces neutralizing HIS, while the inbred Lewis rat is incapable of producing perceptible neutralizing antibody in our experimental model.

Plasmodium falciparum: assay of antigens and antibodies by means of a solid phase radioimmunoassay with radioiodinated staphylococcal protein A.

Human red blood cells (RBC) infected in vitro with Plasmodium falciparum were employed to prepare several types of antigens (sonicated, infected RBC and purified, sonicated merozoites and schizonts). These antigens, as well as control preparations derived from non-infected RBC, were used to coat plastic tubes, which were subsequently tested for capacity to bind anti-P. falciparum antibodies. Binding was detected by means of radio-iodinated staphylococcus protein A. Sera from patients with recent disease or patients who had a history of P. falciparum infection gave strong binding, while sera of normal individuals had only a low binding activity. Some of the antibodies in the positive sera were directed against RBC, since they could bind to tubes coated with normal RBC antigens and could be removed by absorption with RBC. The specificity of the P. falciparum antibodies was confirmed by inhibition tests: preparations derived from infected blood but not from normal blood inhibited the binding activity of the positive sera, to antigen coated tubes.

The role of superoxide dismutation in malaria parasites.

Oxidant stress is associated with the generation of reactive oxygen species that are responsible for the damage of a variety of cellular components. The prevention of such biological damage can be achieved by dismutation of superoxide to H2O2 which in turn is removed by catalase and GSH peroxidase. However, redox-active iron released during the development of plasmodia in the erythrocyte can mediate the conversion of H2O2 to hydroxyl radical which is more reactive. The roles of SOD and the nitroxide SOD mimic 4-OH,2,2,6,6,tetramethyl piperidine-N-oxyl (Tempol) were examined in P. falciparum grown in vitro. Both compounds did not prevent the interference with growth inflicted by various inducers of oxidant stress. Moreover, Tempol inhibited parasite growth, in agreement with previous experiments depicting accelerated mortality in SOD overexpressing mouse model of malaria. Probably, effective defense against ROS requires balanced increments in antioxidant enzymes and is not necessarily improved by an increase in the activity of one enzyme.

Malaria antibodies and mefloquine levels among United Nations troops in Angola.

BACKGROUND: The United Nations deployed about 8,000 soldiers in a peacekeeping mission in Angola. Malaria is the most common disease there and consequently it was the major risk to the UN troops. Most of them are from malaria free areas. As a result of improper prophylactic measures there were many cases of malaria, including some deaths in 1995. In February-March 1996, an Israeli team was sent to Angola to evaluate the malaria situation among UN soldiers. This paper deals specifically with some aspects of chemoprophylaxis and diagnosis. The efforts were concentrated in one particular area where malaria incidence had been reported as the highest. METHODS: Blood samples were collected from nonimmune soldiers who were using mefloquine as a prophylactic drug and were exposed to malaria. The mefloquine and the antimalarial antibody plasma levels were monitored. RESULTS: While the local laboratory indicated that about 80% had a malaria episode, the serological results revealed that only 5 soldiers of the 56 (9%) examined had antimalarial antibodies, of which 3 were Angolans. Despite a controlled prophylactic regimen there was considerable variability in mefloquine plasma levels: 46% of the samples were below the required prophylactic level and 26% above it. All patients who were proven positive with malaria by both microscopic and serologic observation had a low level of mefloquine. CONCLUSIONS: In field conditions, a kit which identifies plasmodial antigens, is preferable, to a microscopic diagnostic method. Controlled mefloquine prophylaxis may not prevent malaria, especially when blood levels are low. The reason for the low mefloquine blood levels is not clear and needs further evaluation.