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.

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.

Biocompatibility of a polymeric implant for the treatment of osteomyelitis.

We evaluated the biocompatibility of an injectable gelling polymeric device for the controlled release of gentamicin sulfate in the treatment of invasive bacterial infections in bone of male Wister rats. The biodegradable delivery carrier, poly(sebacic-co-ricinoleic-ester-anhydride), designated as p(SA:RA), was injected, with and without gentamicin, into the tibial canal. Rats were killed 3 weeks later. The tibiae were processed histologically, leaving the injectable polymer in situ. The local tissue reaction to the polymer with or without antibiotic consisted mainly of mild reactive fibroplasia/fibrosis and mild to moderate increased reactive bone formation. At this stage, no evidence for any active inflammatory response to the polymer was seen. Thus, the injection of p(SA:RA) was well tolerated and did not induce any signs of a progressive inflammatory reaction.

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.

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.

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.

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.

Efficacious treatment of experimental leishmaniasis with amphotericin B-arabinogalactan water-soluble derivatives.

In this study, we tested the efficacy of amphotericin B (AmB)-arabinogalactan (AmB-AG) conjugates for the treatment of experimental leishmaniasis. Chemical conjugation of AmB to a water-soluble, biodegradable, and biocompatible polymer could present many advantages over presently available AmB formulations. Two conjugates were tested, a reduced (rAmB-AG) form and an unreduced (uAmB-AG) form. In vitro, the drug concentrations which lower the values of parasites (for promastigotes) or infected macrophages (for amastigotes) to 50% of the untreated values (ED(50)s) of uAmB-AG and rAmB-AG were 0.19 and 0.34 microg/ml, respectively, for Leishmania major promastigotes and 0.17 and 0.31 microg/ml, respectively, for amastigotes. The effect on Leishmania infantum-infected macrophages was more marked, with ED(50)s of 0.035 microg/ml for rAmB-AG and 0.027 microg/ml for uAmB-AG. In in vivo experiments, BALB/c mice injected with L. major were treated from day 2 onwards on alternate days for 2 weeks. Both conjugates, as well as liposomal AmB (all at 6 mg/kg of body weight) and Fungizone (1 mg/kg), significantly delayed the appearance of lesions compared to that in untreated mice. In addition, both conjugates, but not liposomal AmB, were significantly more effective than Fungizone. Subcutaneous injection of the conjugates (6 mg/kg) was significantly more effective than liposomal AmB in delaying the appearance of lesions. Higher AmB concentrations of up to 12 mg/kg could be administered by this route. When an established infection was treated, uAmB-AG was somewhat more effective than liposomal AmB. In summary, water-soluble polymeric AmB derivatives were found effective and safe for the treatment of leishmanial infections. The conjugates, which are stable and can be produced relatively cheaply (compared to lipid formulations), can be used in the future for the treatment of leishmaniasis infections.

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.

Redox metabolism in glucose-6-phosphate dehydrogenase deficient erythrocytes and its relation to antimalarial chemotherapy.

Experiments in glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes parasitized by Plasmodium falciparum proved that depletion of glutathione increased fluxes of reactive oxygen species and was detrimental to the parasite at various sites and developmental stages. Chloroquine is also considered an inducer of oxidant damage due to its role in preventing heme polymerization. Recently it has been found that GSH prevents cellular damage by degrading the toxic heme. Consequently, we suggest that the use of combinations of chloroquine and depletors of GSH would be highly efficient for the chemotherapy of malaria.

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.

The treatment of animal models of malaria with iron chelators by use of a novel polymeric device for slow drug release.

The hydrophilic desferrioxamine (DFO) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (SIH) are iron chelators which inhibit in vitro proliferation of Plasmodium falciparum with similar potency (IC50 approximately 20 microM in 24- to 48-h tests). The in vivo assessment of these drugs was performed on Swiss mice infected with Plasmodium vinckei petteri with novel modes of drug administration and release. The drugs were delivered postpatently either by multiple i.p. injections or by a single i.p. or s.c. insertion of a drug-containing polymeric device which released most of the drug within 7 days at apparently first-order rates. A regimen of three daily i.p injections of 5 mg DFO for 3 consecutive days or a 70-mg dose of the drug given as an i.p. or s.c. polymer implant evoked similar delay and reduction in peak parasitemias and reduced mortality with no apparent signs of toxicity. Relatively faster, but otherwise similar results were obtained with the less hydrophilic SIH. In combination, the two drugs apparently potentiated each other. The polymeric devices were particularly useful for treating Plasmodium berghei K173-infected C57Bl mice, a suggested model of cerebral malaria, in which classical methods of DFO delivery were ineffective. The insertion of a 140-mg DFO-containing device on day 6 postinfection (parasitemia approximately 1%) led to a marked reduction in parasite proliferation, appearance of neurological sequelae and mortality of mice. Our studies indicate that polymeric devices for slow drug release might be highly advantageous for both hydrophilic and lipophilic drugs whose antimalarial efficacy might depend on the maintenance of sustained blood levels. The results obtained with slow-release devices have implications for malaria chemotherapy as well as for iron chelation therapy in iron overload conditions.

An overview of the malaria situation in Zanzibar.

Zanzibar is composed of two neighbouring islands with about a 1.1 million inhabitants in 2600 square kilometers. Both islands are located off the northeastern coast of the Tanzanian mainland. Zanzibar is a typical malaria endemic area. About a third of all blood samples currently examined for malaria in Zanzibar are positive to Plasmodium falciparum. Previous attempts failed to significantly improve the malaria situation in Zanzibar due to administrative problems, antimalarial drug and DDT resistance. We visited Zanzibar in order to evaluate the magnitude of the malaria problem and the medical logistics. This paper reviews the results of the visit, past and present attempts to eliminate malaria and our conclusions regarding Zanzibar as a model site for attempts to eradicate malaria.

Iron chelators: mode of action as antimalarials.

Malaria parasites growing inside human erythrocytes differ from mammalian cells in their mode of acquisition of bioavailable iron and in their susceptibility to the antiproliferative action of iron chelators. We have assessed here three major properties associated with these phenomena: (a) the stage-dependent nature of parasite iron mobilization from the host and its integration into parasite proteins; (b) the differential permeability of the plasma membrane to iron chelators, and (c) the in situ generation of toxic chelator-metal complexes in the intracellular milieu of infected cells. We have used a combination of synthetic and natural iron chelators with similar iron-binding properties but markedly different capacities to permeate membranes. The profiles of action of these agents on the in vitro growth of Plasmodium falciparum were assessed in terms of inhibitory concentrations, speed of action, stage dependence and reversibility of effects. These profiles provided the basis for a working model of chelator action on parasitized cells. The model allowed us to predict major improvements in the antimalarial performance of iron chelators when used in appropriate combinations of slow-and fast-permeating substances. The synergistic actions found in vitro for various combinations of iron chelators are in accordance with the model and have implications for the design of therapeutic schemes.

Effects of zinc-desferrioxamine on Plasmodium falciparum in culture.

The zinc-desferrioxamine (Zn-DFO) complex is considered to be more permeative into parasitized erythrocytes than is the metal-free DFO. The former may penetrate the cell and exchange its bound zinc for ferric ions, rendering the iron unavailable for vital parasite functions. The effects of these compounds on the in vitro development of Plasmodium falciparum are compared. The results indicate that Zn-DFO is superior to DFO, especially at concentrations below 20 microM, as shown by decreased levels of hypoxanthine incorporation, lower levels of parasitemia, and interference with the life cycle of the parasite. At low concentrations, DFO even enhanced parasite growth. Such an enhancement was not observed following exposure to Zn-DFO. Experiments in which the compounds were removed from the cultures indicated that parasites treated with Zn-DFO are less likely to recover at a later stage. Since DFO has already been used in humans for the treatment of malaria, its complex with zinc, which is more effective in vitro, should also be examined in vivo.

Mode of action of iron(III) chelators as antimalarials. III. Overadditive effects in the combined action of hydroxamate-based agents on in vitro growth of Plasmodium falciparum.

Hydroxamate-based iron(III) chelators exhibit potent antimalarial effects on the asexual stages of Plasmodium falciparum grown in vitro. Antimalarial activity varies with the parasite growth stage and the drug permeation properties. The hydrophilic drug desferrioxamine (DFO) is ineffective on early stages (ring forms) of the parasite due to its poor permeability but irreversibly blocks the growth of advanced stages of parasites. On the other hand, hydrophobic reversed siderophores (RSFs) are more membrane permeable and affect all parasite developmental stages; they affect ring forms irreversibly and trophozoite/schizont forms reversibly and at relatively faster rates, compared with DFO. These observations have provided the basis for postulating a possible overadditive action of the two, distinctly acting, iron chelator types for enhanced antimalarial activity. This was assessed in this study by using novel fast-acting chelators such as RSF derivatives (RSFleum2 and RSFm2) in combination with the relatively slow-acting DFO. Parasite growth was assessed in terms of nucleic acid synthesis and parasitemia. The results indicate that, at any molar ratios of the two types of drugs, the combined inhibitory effect was faster and more potent than the sum of individual effects. The combined drug action showed neither additive nor independent but overadditive properties, as well as sustained inhibition even after drug removal. The potentiating action of RSFs on the long-lasting effects of DFO on parasite growth conformed with the postulated mechanistic model of iron chelator action and iron handling by parasites. Iron chelator combinations might be of therapeutic value.

Antimalarial action of hydroxamate-based iron chelators and potentiation of desferrioxamine action by reversed siderophores.

Hydroxamate-based chelators of iron are potent inhibitors of in vitro growth of Plasmodium falciparum. Two types of such chelators, the natural desferrioxamine and the synthetic reversed siderophore RSFileum2, are prototypes of antimalarial agents whose action spectra differ in the speed of action, stage dependence, and degree of reversibility of effects. This work explores the possibility of improving the antimalarial efficacy of these agents by using them in various combinations on in vitro cultures of P. falciparum. Growth assessment was based both on total nucleic acid synthesis and on parasitemia. The results indicate that the synthetic reversed siderophore more than complements the antimalarial action of desferrioxamine when applied during either ring, trophozoite, or mixed stages. The combined drug effects were significantly higher than the additive effect of the individual drugs. Qualitatively similar results were obtained for both reversible effects and irreversible (i.e., sustained) effects. Following an 8-h window of exposure the combined drug treatment caused parasite growth arrest and prevented its recovery, even 3 days after the treatment. The fact that such a combination of iron chelators displays a wider action spectrum than either drug alone has implications for the design of chemotherapy regimens.