Sporozoites of Mammalian Malaria: Attachment To, Interiorization and Fate Within Macrophages

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozoites into the macrophages does occur.     

Plasmodium berghei: relative immunogenicity of infected reticulocytes and infected oxyphilic red blood cells

Hyperbleeding of mice 1 day before and 1 day after infection with Plasmodium berghei resulted in a more aggravated infection. Parasitemia rose significantly faster, but the mean survival time of these mice was not significantly different from control mice. At Day 5 of infection, parasites were almost exclusively in reticulocytes in contrast to control infections in which parasites were found in oxyphilic erythrocytes at Day 5 after infection. Purified parasitized reticulocytes taken from hyperbled mice at Day 5 after infection contained more young developmental parasite stages than purified parasitized oxyphilic erythrocytes taken from normal mice at Day 5 to 7 after infection. Parasitized reticulocytes were more readily opsonized by antibodies from immune serum when compared to parasitized oxyphilic red blood cells and when used to stimulate immune spleen cells the former were better stimulator cells than the latter. Results suggest either that parasitized reticulocytes are more immunogenic then parasitized oxyphilic red blood cells or that suspensions of parasitized reticulocytes contain more immunogenic parasite stages than suspensions of parasitized oxyphilic red blood cells.     

Comparative study of the phagocytosis of sheep erythrocytes by the macrophages of inbred mice differing in their sensitivity to immunogenic stimulation by the given antigen

The phagocytosis of 14C-labeled sheep red blood cells (SRBC) by the macrophages of isogeneic CBA, BALB/c and C57BL/6 mice was studied. In the presence of bovine serum the macrophages of CBA mice were found to ingest SRBC significantly less actively than the macrophages of BALB/c and C57BL/6 mice. In the presence of isologous serum the macrophages of mice belonging to the strains under study showed quite comparable characteristics with respect to their capacity of ingesting SRBC. The duration of the intracellular digestion of SRBC by the macrophages of mice of these strains did not vary in different strains irrespective of the type of serum.     

A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Sporozoites of mammalian malaria: attachment to, interiorization and fate within macrophages

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozites into the macrohages does occur. Antibodies present in the serum of sporozoite-immunized mice are important in determining the fate of both the intracellular sporozoites and the macrophages containing the parasite. Sporozoites coated with antibodies degenerate within vacuoles of the macrophages, which have no morphologic alteration. Sporozoites incubated in normal serum do not degenerate within macrophages, but the parasitized macrophages become morphologically altered and are destroyed. Preliminary experiments indicate that sporozoites appear to interact with rat Kupffer cells in the same way as with the peritoneal mouse macrophages. It is postulated that Kupffer cells play a dual role in sporozoite-host cell interaction. In normal animals these cells might serve to localize the sporozoites in the immediate vicinity of the hepatocytes. In the immunized animals, macrophages would remove and destroy the antibody-coated parasites, thus contributing to sporozoite-induced resistance.     

思连康对小鼠腹腔巨噬细胞吞噬率和吞噬指数的影响

目的 研究双歧杆菌四联活菌片（商品名：思连康）对小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率及吞噬指数的影响。方法 将SPF小鼠30只随机分成三组,每组10只,Ⅰ组灌胃生理盐水,Ⅱ组灌胃婴儿双歧杆菌菌悬液,Ⅲ组灌胃双歧杆菌四联活菌片菌悬液,每天给药0.5 mL,菌液浓度为1.0×108 CFU/mL,连续给药10 d后小鼠腹腔注入2%鸡红细胞悬液1 mL（红细胞数量为2×108个/mL）,30 min后处死,取小鼠腹腔洗液,观察并记录吞噬鸡红细胞的巨噬细胞数及被吞噬的鸡红细胞数,计算吞噬率及吞噬指数。结果 与Ⅰ组相比,Ⅱ组和Ⅲ组小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数均显著升高（Ps＜0.05）,其中Ⅲ组高于Ⅱ组（P＜0.05）。结论 双歧杆菌四联活菌片及其婴儿双歧杆菌通过提高小鼠腹腔巨噬细胞的吞噬率和吞噬指数提高机体的免疫力。     

Hepcidin is regulated during blood-stage malaria and plays a protective role in malaria infection

Hepcidin is one of the regulators of iron metabolism. The expression of hepcidin is induced in spleens and livers of mice infected with pathogenic bacteria. Recent studies have indicated that serum hepcidin level is also increased in human subjects infected with Plasmodium falciparum. The mechanism of the regulation of hepcidin expression and its role in the infection of malaria remains unknown. In this study, we determined the expression of hepcidin in livers of mice infected with Plasmodium berghei. The expression of hepcidin in the liver was upregulated and downregulated during the early and late stages of malaria infection, respectively. Inflammation and erythropoietin, rather than the iron-sensing pathway, are involved in the regulation of hepcidin expression in livers of infected mice. Meanwhile, we investigated the effect of hepcidin on the survival of mice infected with P. berghei. Treatment of malaria-infected mice with anti-hepcidin neutralizing Abs promoted the rates of parasitemia and mortality. In contrast, lentiviral vector-mediated overexpression of hepcidin improved the outcome of P. berghei infection in mice. Our data demonstrate an important role of hepcidin in modulating the course and outcome of blood-stage malaria.     

Haptoglobin and malaria

Haptoglobin gene knockout mice and wild-type controls were infected with Plasmodium berghei ANKA or Plasmodium chabaudi. The peak parasitaemia and parasite burden were higher in Hp-/- mice than in Hp+/+ mice. The increase in spleen weight following malaria infection was smaller in Hp-/- mice than in Hp+/+ animals. The occurrence of cerebral malaria in P. berghei ANKA infection was not different in Hp gene knockout mice and their controls.     

Protection of mice against plasmodium and babesia infections: attempts to raise host-protective sera.

In an attempt to generate large numbers of mice resistant to Plasmodium berghei and Babesia rodhaini to be used as donors of antibody-secreting cells for hybridoma production, various methods of inducing resistance to repeated challenge with infected blood cells have been explored. Although results of independent experiments varied markedly, prior injection of CBA/M mice with BCG, and prior infection of BALB/c mice with Plasmodium yoelii, were found to be manipulations capable of inducing resistance to P. berghei. A single dose of serum, harvested from resistant mice challenged several times with P. berghei, could transfer resistance against P. berghei to a proportion of naive CBA/H recipients. Although resistance to multiple B. rodhaini challenge could be induced in mice, in no situation was a host protective effect of a single high dose of serum demonstrated in naive recipients.     

Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro.

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.     

Modulation of the Host's Immune Response to Plasmodium berghei by a Parasite-Derived Immunosuppressive Factor

ABSTRACT. It is well known that Plasmodium -infected hosts are immunosuppressed, as shown by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei -infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Comparative studies on the infectivity of Plasmodium berghei gametocytes and ookinetes for gnotobiotic and xenobiotic Anopheles stephensi

Previous studies indicated that gnotobiotic Anopheles stephensi mosquitoes were less susceptible to infection with Plasmodium berghei than xenobiotic ones (Munderloh and Kurtti, 1985). Groups of 100 to 200 mosquitoes were fed on infected hamsters, heparinized gametocytemic blood (via a membrane feeder), and in vitro-formed ookinetes suspended in blood (membrane feeder). Xenobiotic A. stephensi were readily infected by all 3 routes. Gnotobiotic mosquitoes consistently acquired infection after engorging on hamsters (average level of infected females in 8 experiments: 54.1%), but the parasite yield was low (average number of oocysts per infected female: 21.6). In 7 experiments where gnotobiotic A. stephensi were membrane-fed infected hamster blood, an average of only 8.8% of the females became infected, harboring a mean of 2.4 oocysts, and in 7 additional cases no infection was achieved. This pattern was reversed when gnotobiotic A. stephensi were fed ookinetes. A larger proportion of them became infected (mean level of infection in 8 experiments: 76.2%) and they acquired a higher mean number of oocysts per female (94.4) than did xenobiotic mosquitoes. Thus, gnotobiotic A. stephensi are as able as xenobiotic ones to support the sporogonic development of P. berghei, but are less able to support ookinete development.     

Induction of macrophage motility by a T-cell line from Balb/c mice specific for Plasmodium berghei malaria

A long-term antimalaria T-cell line (AMTL) expressing a helper phenotype (Thy 1.2+, Lyt 2.2-) was established from Plasmodium berghei-recovered Balb/c mice. The ability of this T-line to induce macrophage motility was measured in vivo and in vitro. Adoptive transfer of AMTL cells to normal Balb/c mice showed an increased delayed hypersensitivity response to the homologous antigen, i.e., parasitized erythrocytes (PE). In vitro, AMTL culture supernatant (AMTL-SUP) augmented chemotactic locomotion of macrophages derived from both normal and infected mice. However, the effect on normal macrophages was significantly higher. AMTL cells adoptively transferred to normal mice had no effect on parasitemia levels or mortality rate after subsequent infection with P. berghei. Partial characterization of the AMTL-SUP indicated the involvement of a protein of about 12,600 Daltons in the enhancement of chemotaxis. These findings suggest that the AMTL cells and chemoattractants produced by them can induce macrophage motility, and that the macrophage malfunction in Balb/c with P. berghei infection is not due to defects at the T-lymphocyte level.     

Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes

Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. ? 2011 International Society for Advancement of Cytometry.     

The liver stage of Plasmodium berghei inhibits host cell apoptosis

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite-host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei-infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei-infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-alpha/D-galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-alpha-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.     

Characterization of the nonphagocytic adherent cell from the peritoneal cavity of normal and BCG-treated mice.

A distinctive subpopulation of nonphagocytic, tightly adherent cells (NPAC) comprised approximately 6% of the adherent peritoneal cells from untreated mice, and about 18% of those from mice previously given BCG i.p. A separation procedure based on adherence and lack of phagocytosis was devised. Isolated NPAC were morphologically intermediate between small lymphocytes and macrophages. They were positive for nonspecific esterase, negative for peroxidase, positive for surface IgM, and negative for surface IgG1, IgG2 and IgA. When capped, their surface IgM regenerated in vitro. NPAC had demonstrable Fc receptors but not EAC receptors. They resisted killing by an anti-macrophage serum, were negative by immunofluorescence with an anti-T cell reagent, and incorporated increased amounts of thymidine in response to LPS but not to PHA. They were more readily killed with anti-Ia serum and complement than macrophages, but less readily than splenic B cells. NPAC appeared to represent a subpopulation of B lymphocytes which contaminates some preparations previously regarded as "macrophages" and which may be ressponsible for some of the activities previously ascribed to "macrophages".     

Iron deficiency influences the course of malaria in Plasmodium berghei infected mice

Iron deficiency accelerates suicidal erythrocyte death, which is evident from phosphatidylserine exposure. The present study explored whether iron deficiency compromises intraerythrocytic growth of Plasmodium and enhances death of infected erythrocytes thus influencing the course of malaria. As a result, phosphatidylserine exposure is increased in Plasmodium falciparum infected human erythrocytes, an effect significantly more marked in iron deficiency. Moreover, iron deficiency impairs in vitro intraerythrocytic growth and infection of erythrocytes. In mice, iron-deficient erythrocytes are more rapidly cleared from circulating blood, an effect increased by infection with Plasmodium berghei. Parasitemia in P. berghei infected mice was significantly decreased (from 54% to 33% of circulating erythrocytes 20 days after infection) and mouse survival significantly enhanced (from 0% to 20% 30 days after infection) in iron-deficient mice. In conclusion, iron deficiency favourably influences the course of malaria, an effect partially due to accelerated suicidal death and subsequent clearance of infected erythrocytes.     

Effect of prior eradication of Plasmodium berghei infection on the foetal development and parasitaemic levels under the stress of pregnancy

Pregnant mice infected on gestation day (GD) 13 with Plasmodium berghei had similar rate of parasitaemia and mortality as non pregnant controls. 50% of pregnant infected mice had normal delivery, 20% had absorbed foetuses and 30% died before parturation. However, animals infected with P. berghei, treated with drugs (sulfadiazine or chloroquine) had normal foetal development. No recrudescence occurred in either of these groups of animals even under the stress of pregnancy indicating protection. Pups born to pregnant infected mice (GD) 13 had low body weight as compared to pups born to mice which were infected and treated with drugs. Histopathological findings showed hyperplasia of trophoblast and plugging of placental sinusoids with parasitized erythrocytes in animals infected on GD 13 only. It is felt that prior eradication of malaria infection results in development of sufficient immunity which prevents the mal development of foetuses or parasitaemic levels under the stress of pregnancy.