Plasmodium Berghei-infected Red Blood Research Articles

Comparing Hydroxychloroquine and Pyrimethamine With Sulfadoxine-Pyrimethamine to Treat Plasmodium berghei Infection in Mice

Background: Malaria is caused by a parasitic protozoan called Plasmodium with an estimated mortality rate of one million people annually and it is estimated that half of the world's population are at risk for this disease. Objectives: The current study aimed to compare the effect of hydroxychloroquine and pyrimethamine with sulfadoxine-pyrimethamine to treat Plasmodium berghei infection in mice. Pyrimethamine, hydroxychloroquine and sulfadoxine-pyrimethamine were used in the current study. Methods: Four groups of mice each including nine mice were selected. The mice were infected by intraperitoneally injection of Plasmodium berghei infected red blood cells. One group was considered as a positive control and three other ones received each one of the drugs. On days 4, 7, 14, 21 and 28, a blood smear was prepared from each mouse, stained with Giemsa and the parasitemia rate was calculated. Results: Parasitemia in the positive control group showed an increase on days four and seven at a level of 5.9% and 23.7%, respectively, which was statistically significant compared with the other groups (P < 0.05). The parasitemia remarkably decreased in the group receiving sulfadoxine-pyrimethamine than in the one taking pyrimethamine (P < 0.05). In addition, the mortality rate in mice treated with sulfadoxine-pyrimethamine was lower than those of the other groups (P < 0.05). The survival time in the group treated with sulfadoxine-pyrimethamine was 28 days on average and in the group treated with pyrimethamine and hydroxychloroquine were 15.56 and 26.44 days, respectively. Conclusions: The results of the article suggested that malaria treatment strategy shifts completely from the monotherapy; therefore, the combination drugs should be used for this purpose, especially in endemic areas.

Measuring antigen presentation in mouse brain endothelial cells ex vivo and in vitro.

We have recently demonstrated that brain endothelial cells cross-present parasite antigen during mouse experimental cerebral malaria (ECM). Here we describe a 2-d protocol to detect cross-presentation by isolating the brain microvessels and incubating them with a reporter cell line that expresses lacZ upon detection of the relevant peptide-major histocompatibility complex. After X-gal staining, a typical positive result consists of hundreds of blue spots, compared with fewer than 20 spots from a naive brain. The assay is generalizable to other disease contexts by using reporter cells that express appropriate specific T cell receptors. Also described is the protocol for culturing endothelial cells from brain microvessels isolated from naive mice. After 7-10 d, an in vitro cross-presentation assay can be performed by adding interferon-γ, antigen (e.g., Plasmodium berghei-infected red blood cells) and reporter cells in sequence over 3 d. This is useful for comparing different antigen forms or for probing the effects of various interventions.

Antimalarial and safety evaluation of Pluchea lanceolata (DC.) Oliv. & Hiern: In-vitro and in-vivo study

Ethnopharmacological relevanceMany of the effective therapeutic strategies have been derived from ethnopharmacologically used natural products. Pluchea lanceolata is an herb employed in Indian folk medicine for malaria like fever but it lacks proper pharmacological intervention. Aim of the studyTo evaluate antimalarial and safety profile of Pluchea lanceolata: an in-vitro, in-vivo for its ethnopharmacological validation. Materials and methodsMethanol, butanol, ethyl acetate, chloroform, hexane extracts and its isolate, taraxasterol acetate (TxAc) were obtained from air dried aerial part of Pluchea lanceolata. These were tested in-vitro against chloroquine-sensitive strain of Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase activity. The most potent hexane extract and TxAc were further validated for in-vivo antimalarial and safety evaluation. TxAc, a pentacyclic-triterpene isolated from the most active fraction was further evaluated with special emphasis on inflammatory mediators involved in malaria pathogenesis. Murine malaria was induced by intra-peritoneal injection of Plasmodium berghei infected red blood cells to the male Swiss inbred mice. Mice were orally treated following Peters 4-Day suppression test. In-vivo antimalarial efficacy was examined by evaluating the parasitaemia, percent survival, mean survival time, blood glucose, haemoglobin and pro-inflammatory mediators involved in malaria pathogenesis. ResultsHexane extract and TxAc showed promising antimalarial activity in-vitro and in-vivo condition. TxAc attributed in inhibition of the pro-inflammatory cytokines as well as afford to significant increase in the blood glucose and haemoglobin level when compared with vehicle treated infected mice. We have not observed the synergistic action of combinations of chloroquine and TxAc from our experimental results. In-vitro and in-vivo safety evaluation study revealed that hexane extract is non toxic at higher concentration. ConclusionPresent study further validates the ancient Indian traditional knowledge and use of Pluchea lanceolata as an antimalarial agent. Study confirms the suitability of Pluchea lanceolata as a candidate for further studies to obtain a prototype for antimalarial medicine.

Role of Oxidative Stress and Apoptosis in the Placental Pathology of Plasmodium berghei Infected Mice

Placental malaria is a common clinical complication during pregnancy and is associated with abortion, premature delivery, intrauterine growth retardation and low birth weight. The present study was designed to delineate the underlying mechanism of placental pathology during malarial infection with special reference to oxidative stress and apoptosis. Experimentally, pregnant BALB/c mice were infected with Plasmodium berghei infected red blood cells on gestation day 10. The presence of malarial infection in placenta was confirmed by histopathological studies. It was observation that infected placenta had plugged placental sinusoids with parasitized red blood cells and malarial pigments. Interestingly, we found significant increase in the level of malondialdehyde, the index of oxidative stress and decreased activity of catalase, the antioxidant in infected placenta. Furthermore, in infected placenta the oxidative stress mediated apoptosis was determined by DNA fragmentation assay, ethidium bromide/acridine orange staining and caspase activity. It was observed that oxidative stress begin after second day of malarial infection. Interestingly, it was observed that there was down regulation of anti-apoptotic protein Bcl-2 and up regulation of pro-apoptotic protein Bax in infected placenta, suggesting the involvement of mitochondrial pathway of apoptosis which was further confirmed by activation of caspase 9. However, no change in the expression of Fas gene and caspase 8 activity, indicated the absence of death receptor pathway. Thus, it can be concluded that the placental pathology during malarial infection is mediated by mitochondrial pathway of apoptosis occurring due to augmented lipid peroxidation which may in turn jeopardise the materno-fetal relationship.

Flow cytometric enumeration of Plasmodium berghei-infected red blood cells stained with SYBR Green I

High-throughput methods for evaluation of in vivo efficacy of candidate compounds against Plasmodium parasites are necessary during the antimalarial drug development process. It is essential that enumeration of parasitemia in the infected blood from experimental host animals is accurate and reliable. Flow cytometric enumeration of parasitized cells stained with fluorescent dye is a rapid alternative method to conventional microscopic counting. In this study, a protocol for flow cytometric enumeration of rodent malaria parasite Plasmodium berghei-infected red blood cells (RBC) stained with SYBR Green I was developed. The optimal concentration of SYBR Green I used to stain infected RBC was 4× for 30min. This SYBR Green I staining protocol in combination with the bi-dimensional FL-1530/FL-3620 detection method accurately detects parasitemia above 0.02%. The dye is stable during the prolonged incubation period necessary for accurate enumeration of parasitemia, with no loss of fluorescent signal over a period of hours. This protocol was validated in an antimalarial assay and the result was comparable to that obtained from conventional microscopic counting. The SYBR Green I flow cytometric protocol is thus a rapid and precise tool for high-throughput in vivo antimalarial drug screening.

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites.

Three monoclonal antibodies generated by immunization of mice with Plasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and erythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365-681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites of P. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity of P. berghei sporozoites and to inhibit the development of liver stages of P. yoelii.

Stage-specific proteins of Plasmodium berghei-infected red blood cells detected by antibodies of immune mouse serum.

The asynchronously developing malaria parasite Plasmodium berghei was synchronized using in vitro cultivation techniques (Mons et al. 1985). After the infection of naive mice, preparations of parasitized erythrocytes with a high level of synchronism could be obtained. Immunofluorescence and immunoblotting techniques using serum from immunized mice were applied to determine stage-specific immunogenic molecules in the parasitized erythrocyte preparations. These techniques allowed the detection of not only parasite-derived but also altered-self molecules. Membrane fluorescence of infected erythrocytes was detected only in preparations containing late trophozoites and schizonts. The appearance of this fluorescence pattern coincided with the presence of immunogenic polypeptides of mol. wt. greater than 200 kD, 86 kD, and 56 kD especially, among some other polypeptides. Preliminary experiments using lactoperoxidase-catalyzed radioiodination suggested that the greater than 200 kD and 56 kD molecules were present at the erythrocyte surface. One molecule with mol. wt. 153 kD was associated with the presence of ring-infected erythrocytes. However, membrane fluorescence of ring-stage-infected erythrocytes was not found. Noninfected erythrocytes sometimes showed membrane fluorescence.

Serum Opsonic Activity in Rodent Malaria: Functional and Immunochemical Characteristics in Vitro

Abstract The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.

Plasmodium berghei: Polyribosome profiles in the spleens of infected mice

The distribution pattern of splenic polyribosomes in sucrose gradients was investigated in Swiss mice which had been inoculated with Plasmodium berghei-infected red blood cells. The onset of polyribosome formation in the spleen appeared to coincide with the first peak of parasitemia and to parallel the increasing pyroninophilia of the proliferating erythropoietic tissue. The polysome profile appeared to be similar after induction of anemia by phenylhydrazine and differs completely from the biphase profile which is obtained after antigenic stimulation with sheep red blood cells. The results suggest that the spleen is not involved in the synthesis of antibodies against a parasitic antigen during the course of a primary lethal infection with Plasmodium berghei.

Plasmodium berghei Infection in Neonatally Thymectomized Hamsters

The effect of neonatal thymectomy on immunity to Plasmodium berghei in the golden hamster was studied in 43 hamsters. Thymectomized, sham-operated, and unoperated controls were infected at 4 and 8 weeks of age by intraperitoneal injection of 15 X 106 parasitized hamster red blood cells. Parasitemias were estimated from Giemsa-stained blood films taken at 2to 3-day intervals after infection. Neonatally thymectomized hamsters developed parasitemias more rapidly and had earlier and higher mortality than did the sham-operated or unoperated controls. These observations suggest that thymic-dependent cell-mediated immunity plays a major role in acquired immunity to P. berghei in the golden hamster. The importance of the thymus in the development of immunologic competence in a variety of animals is well established (Miller, 1964; Good and Papermaster, 1964). Neonatal thymectomy impairs the development of immune capability in many animal species including certain rodents such as hamsters (Sherman, Adner, and Dameshek, 1964), rats (Arnason, Jankovic, and Waksmann, 1962), and mice (Miller, 1961). The importance of thymus-dependent immune mechanisms in plasmodial infections is not so well known. Brown, Allison, and Taylor (1968), Stechschulte (1969), and Hanson and Chapman (unpublished) noted increased parasitemias and increased mortality in neonatally thymectomized rats challenged with P. berghei as compared to sham-operated or unoperated controls. Plasmodium berghei infections produce approximately 100% mortality in young hamsters (Adler, 1954; Nussenzweig et al., 1966; Jarvis, MaCallum, and Sprinz, 1968) while some hamsters infected with P. berghei at an older age recover from the infection and are immune to superinfection (Adler, 1954). Wright (1968) reported that neonatally thymectomized hamsters infected with Plasmodium berghei survived significantly longer than sham-operated or unoperated controls. Preliminary results in our laboratory indicated that neonatally thymectomized hamsters were more susceptible to P. berghei than controls. These studies were undertaken to extend our preliminary investigations on the effect of neonatal thymectomy on Received for publication 14 May 1970. 24 the immune response of hamsters to P. berghei infection. MATERIALS AND METHODS Random-bred pregnant Syrian hamsters (Mesocricetus auratus) were obtained from Lakeview Hamster Colony, Newfield, N. J., and maintained in our laboratory until parturition. Neonatal hamsters were thymectomized within 30 hr of birth. The hamsters were anesthetized with ether and immobilized on a small surgical board. The sternum was split with small scissors at the left paramedian from thoracic inlet to the 4th rib. The surgical board was then placed on a stereoscopic microscope and the small right and left thymus were removed by blunt dissection. The board was immediately removed from the dissecting microscope and the incision was closed with 2 to 3 simple interrupted sutures of 6-0 silk. The thorax of sham-operated hamsters was incised and sutured in similar manner. The neonates were placed at 37 C for 1 to 2 hr for surgical recovery and subsequently cleaned and returned to the mother. Neonates were handled with surgical gloves throughout the surgery to decrease cannibalism. Approximately 70% survival from surgery was obtained in some litters. Cannibalism was a problem in many instances. Plasmodium berghei infected red blood cells (RBC) were taken from rats and the parasites were adapted to hamsters by 2 or 3 passages at 10to 12-day intervals. Thymectomized, sham-operated, and unoperated hamsters of equivalent age including individuals of both sexes were infected with 15 X 106 parasitized red blood cells by intraperitoneal (I.P.) injection. Blood smears were taken from the tail at various intervals, stained with Giemsa's stain, and numbers of parasites were estimated according to the method of Marshall, Litchfield, and White (1942) as modified by Thompson, Bayles, and Olszewski (1970). Reticulocytes were counted as described by Ott (1968). Blood samples were collected in microhematocrit tubes for packed cell volume (PCV) determinaThis content downloaded from 157.55.39.171 on Sat, 25 Jun 2016 07:09:41 UTC All use subject to http://about.jstor.org/terms CHAPMAN AND HANSON-PLASM. BERGHEI IN THYMECTOMIZED HAMSTERS 25