Production of interferon-gamma during infection of mice with Plasmodium chabaudi chabaudi

An ELISA assay, designed to detect interferon-gamma (IFN-gamma) in the picogram range, was used to study the presence of IFN-gamma in serum and its production by T cells taken from C57BL/6 mice infected with Plasmodium chabaudi chabaudi. IFN-gamma was detectable in mouse plasma for two to three days before the peak of parasitaemia. Similarly, IFN-gamma production by T cells could be detected in vitro. In limiting dilution cultures, the production of IFN-gamma by as few as 1,000 T cells was detectable using this assay. The limiting dilution analysis revealed that a substantial IFN-gamma response by specific T cells occurs very early in a primary infection with P. chabaudi.     

Iron deficiency protects inbred mice against infection with Plasmodium chabaudi.

Plasmodium chabaudi infections of NFR/N mice made anemic by dietary iron deficiency produced mortalities of 25% (male) and 7% (female) compared with 100% in iron-sufficient controls. When iron-deficient mice convalescing from the primary infection were returned to the normal diet, 100% experienced recrudescent parasitemia. No recrudescence occurred in mice maintained on the iron-deficient diet.     

Role of gamma interferon during infection with Plasmodium chabaudi chabaudi.

A role has been proposed for inflammatory mediators such as gamma interferon (IFN-gamma) and reactive oxygen intermediates in the control of the blood stages of Plasmodium organisms. It was previously shown that IFN-gamma can be detected in the plasma of mice with a primary infection by Plasmodium chabaudi chabaudi (AS). We found that susceptible and other resistant mouse strains produced IFN-gamma, suggesting that susceptibility is not due to a defect in IFN-gamma production. Administration of IFN-gamma to intact C57BL/6 mice slightly decreased and partially delayed parasitemia, whereas in vivo depletion of IFN-gamma through injection of a "cocktail" of monoclonal antibodies against IFN-gamma exacerbated infection. Since CD4+ T cells are essential for the development of a protective immune response to P. chabaudi chabaudi, we tested whether CD4+ T cells are responsible for IFN-gamma production in vivo and whether exogenous IFN-gamma can replace the protective function of the CD4+ T cells. Mice depleted of CD4+ T cells were unable to produce IFN-gamma, but factors in addition to IFN-gamma may be important in parasite clearance.     

Reconstitution of B-cell-depleted mice with B cells restores Th2-type immune responses during Plasmodium chabaudi chabaudi infection.

In mice depleted of B cells from birth by treatment with anti-immunoglobulin M(mu) antibodies, progression from a Th1- to a Th2-regulated immune response during primary infection with Plasmodium chabaudi chabaudi fails to occur. While Th1-type immunity limits parasitemia, in the absence of B cells, chronic low-grade infections persist. Here, we show that reconstituting immune, and to a lesser extent naive, B cells to mice rendered deficient in B-cell function through anti-immunoglobulin M(mu) pretreatment restores the CD4+ T-cell response to the Th2 type later in P. c. chabaudi infection and with it the capacity to eliminate infection. This finding provides clear evidence that B cells are required for switching the balance of immune regulation between CD4+ T cells from Th1 to Th2 during P.c. chabaudi infection and supports the concept that B cells, through antibody production, are needed for effective antimalarial immunity.     

Blood transfusion alters the course and outcome of Plasmodium chabaudi AS infection in mice.

The importance of severe anemia in the mortality of susceptible A/J mice during blood-stage Plasmodium chabaudi AS infection was assessed. Blood transfusion during and 2 to 3 days after peak parasitemia rescued 90% of susceptible mice from severe anemia and death and allowed these mice to clear the infection and acquire immunity to reinfection. However, blood transfusion prolonged the patency of the infection for up to 5 days after peak parasitemia. Blood transfusions in resistant C57BL/6 mice produced an identical effect, that is, prolongation of the patency of parasitemia. In addition, blood transfusion increased the numbers of gametocytes in both mouse strains. In both strains of mice, the rapid reduction in parasitemia, which occurs during crisis, was associated with the development of moderate levels of anemia. The possible mechanisms for the modulation of parasitemia by blood transfusion and the implications of the present observations for our understanding of the events which occur during crisis are discussed. It is proposed that parasitologic crisis is induced and/or maintained by physiological alterations associated with anemia.     

Osteopontin participates in Th1-mediated host resistance against nonlethal malaria parasite Plasmodium chabaudi chabaudi infection in mice

Osteopontin (OPN) knockout mice (OPN-KO mice) died of Plasmodium chabaudi chabaudi infection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.     

Clonal analysis of B lymphocyte responses to Plasmodium chabaudi infection of normal and immunoprotected mice.

Parasite infection causes marked perturbations in the host immune system, as shown by hypergammaglobulinemia, autoimmunity and immune depression, but there is little information on the number, specificities and performance of B cell clones activated in the course of infection. We have addressed these questions in a model of murine malaria induced by Plasmodium chabaudi, where primary infection results in very marked B cell responses that shift in Ig isotype pattern in immunoprotected animals, and where immunity can be transferred to naive recipients by injection of serum from late, but not early, infection. We have quantitated B cells responding to infection in two distinct functional compartments, namely blast cells and Ig-secreting cells, and compared normal with immune animals. We have also determined the frequencies of clonal specificities towards several autoantigens (DNA, myosin, transferrin and red cells), non-self protein or polysaccharide antigens (KLH, levan and dextran), and parasite antigens in both compartments, by measuring blast cell reactivities in limiting dilution analyses and Ig secretion in ELISASPOT assays. This experimental design allowed us to assess the specificity of the B cell responses, to compare the clonal composition of these two B cell compartments, and to evaluate putative specific response regulation at the step of terminal differentiation. Our results show that, in this particular experimental system: (i) B cell responses in primary infection are truly non-specific while immune animals show a greater ability to control the massive non-specific response; (ii) parasite specific B cells, particularly those committed to IgG production, are selectively stimulated in immune individuals; (iii) autoreactive B cells are not selectively stimulated, but increased autoantibody production may result from perturbation in the control of terminal differentiation in the respective clones; (iv) clones with specificity to some non-self antigens (e.g. KLH and dextran) are selectively engaged and regulated, which might have implications for the immunosuppression following infection.     

Protective effect of severe magnesium deficiency on Plasmodium chabaudi infection

Mice were fed for 30 days on purified diets containing 50 (severely Mg deficient diet), 100 (moderately Mg deficient diet) and 1300 mg/kg (control diet). An additional group raised on stock UAR diet was also used for the experiment. The mice were maintained on the experimental diets for 12 days before being inoculated with P. chabaudi. Infection evolved similarly in mice fed the control purified diet, moderately Mg deficient diet and the stock diet whereas the severely Mg deficient diet induced a 50% decrease in malarial infection as shown by the decrease in the percentage of parasitized red blood cells (RBC). In control mice, RBC Mg values increased significantly during P. chabaudi infection; however RBC Mg values were significantly lower in Mg-deficient than in control animals.     

Transforming growth factor beta-induced failure of resistance to infection with blood-stage Plasmodium chabaudi in mice

The role of transforming growth factor beta (TGF-beta) in infection with Plasmodium chabaudi was investigated with resistant and susceptible mouse models. C57BL/10 mice produced gamma interferon (IFN-gamma) and nitric oxide (NO) shortly after infection and cleared the parasite spontaneously. In contrast, BALB/c mice showed a transient enhancement of TGF-beta production, followed by a relative lack of IFN-gamma and NO production, and succumbed to the infection. However, there was no correlation between levels of serum TGF-beta and splenic TGF-beta mRNA in both mouse strains before and after infection. Administration of recombinant TGF-beta (rTGF-beta) rendered resistant mice susceptible because of suppression of subsequent production of IFN-gamma and NO. Administration of anti-TGF-beta antibody to the infected BALB/c mice resulted in remarkable increases in serum IFN-gamma and NO, and the mice resisted the infection. Splenic CD4(+) T and CD11b+ cells of C57BL/10 mice were significantly activated after infection, but this was completely abrogated by administration of rTGF-beta. These results suggested that, in the P. chabaudi-susceptible but not resistant mice, production of TGF-beta was promoted, and subsequent failure of IFN-gamma- and NO-dependent resistance to the parasite was induced. This study is the first to indicate that TGF-beta production was the key event in failure of resistance to mouse malaria.     

Immunization of mice with phosphatidylcholine drastically reduces the parasitaemia of subsequent Plasmodium chabaudi chabaudi blood-stage infections.

It has been suggested that phospholipids and antibodies directed against phospholipids are important in the pathology of malaria. We have investigated the influence of immunizations with phospholipids on the course of subsequent blood-stage Plasmodium chabaudi chabaudi infections in ICR inbred mice. We observed a significant reduction in the parasitaemia following immunization with phosphatidylcholine (PC), but not with phosphatidylethanolamine (PE) immunization. At the peak of the infection, PC-immunized mice displayed a T-helper 2 (Th2)-type cytokine production pattern, whereas PE-immunized or non-treated controls displayed a cytokine production pattern of the T-helper 1 (Th1) type. Serum immunoglobulin transfer from PC-immunized mice protected naive mice in a similar fashion to PC-immunization, demonstrating that the observed reduction of parasitaemia was caused by the presence of PC-specific antibodies.     

Genetic control of susceptibility to infection with Plasmodium chabaudi chabaudi AS in inbred mouse strains

To identify genetic effects modulating the blood stage replication of the malarial parasite, we phenotyped a group of 25 inbred mouse strains for susceptibility to Plasmodium chabaudi chabaudi AS infection (peak parasitemia, survival). A broad spectrum of responses was observed, with strains such as C57BL/6J being the most resistant (low parasitemia, 100% survival) and strains such as NZW/LacJ and C3HeB/FeJ being extremely susceptible (very high parasitemia and uniform lethality). A number of strains showed intermediate phenotypes and gender-specific effects, suggestive of rich genetic diversity in response to malaria in inbred strains. An F2 progeny was generated from SM/J (susceptible) and C57BL/6J (resistant) parental strains, and was phenotyped for susceptibility to P. chabaudi chabaudi AS. A whole-genome scan in these animals identified the Char1 locus (LOD=7.40) on chromosome 9 as a key regulator of parasite density and pointed to a conserved 0.4-Mb haplotype at Char1 that segregates with susceptibility/resistance to infection. In addition, a second locus was detected in [SM/J × C57BL/6J] F2 mice on the X chromosome (LOD=4.26), which was given the temporary designation Char11. These studies identify a conserved role of Char1 in regulating response to malaria in inbred mouse strains, and provide a prioritized 0.4-Mb interval for the search of positional candidates.     

Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.

The induction of T helper cell subsets during the course of non-lethal or lethal blood-stage Plasmodium chabaudi AS infection was investigated using inbred strains of mice which differ in the level of resistance to this intraerythrocytic parasite. Resistant C57Bl/6 mice experience a non-lethal course of infection characterized by moderate levels of both parasitaemia and anaemia and resolution of primary acute infection by 4 weeks, while susceptible A/J mice experience lethal infection with fulminant parasitaemia and severe anaemia. T helper subset function was assessed during infection by determining the kinetics of spleen cell production in vitro of the Th1-derived cytokine, interferon-gamma (IFN-gamma), and of the Th2-derived cytokine, IL-5, using sandwich ELISAs. Spleen cells from resistant C57Bl/6 mice were found to produce high levels of IFN-gamma within 1 week of infection in response to both the mitogen concanavalin A (Con A) and malaria antigen. Furthermore, CD4+ T cells were found to be the source of IFN-gamma while both CD4+ and CD8+ T cells were found to produce IL-5. Decreased IFN-gamma production after day 10 was concomitant with significant production of IL-5 between 2 and 3 weeks post infection. In contrast, spleen cells from susceptible A/J mice produced high levels of IL-5 within the first week of infection. In addition, these animals were found to have high serum levels of IL-5. These results, thus, confirm previous observations that resolution of primary blood-stage P. chabaudi infection occurs by sequential activation of Th1 CD4+ T cells followed by activation of the Th2 subset, and in addition, suggest that induction of a strong Th2 response early in infection may lead to a severe and lethal course of malaria.     

In vivo antimalarial evaluation of MAMA decoction on Plasmodium berghei in mice

The use of decoctions of different plant materials is common practice in antimalarial ethnomedicine in Africa. Scientific evaluation of such herbal combinations to verify the claims is important. The study has evaluated the antimalarial efficacy of MAMA decoction (MD), a multicomponent herbal preparation and its individual plant components, namely leaves of Morinda lucida Benth [Rubiaceae] (ML), Azadirachta indica A. Juss [Meliaceae] (AI), Alstonia boonei De Wild [Apocynaceae] (AB) and Mangifera indica L [Anacardiaceae] (MI) in Plasmodium berghei-infected mice. Each decoction was prepared by boiling the powdered leaf in water, concentrated in vacuo and freeze-dried. The acute toxicity of MD (LD₅₀?=?3.8 g/kg) was determined using Lorke's method. The antimalarial activities of MD and its plant components were evaluated by oral administration of the freeze-dried extracts (15–240 mg/kg) using the early malaria infection test model. The established malaria infection test was used to evaluate MD (60–240 mg/kg) while amodiaquine [10 mg/kg] (AQ) and distilled water were employed as the positive and negative controls, respectively. From the early malaria infection test, the effective doses at 50 % (ED₅₀) and 90 % (ED₉₀) for MD, AB, AI, ML, MI and AQ were 43, 79, 140, 134, 208 and 3.9 mg/kg and 202, 276, 291, 408, 480 and 9.2 mg/kg, respectively. For the established infection test, MD (240 mg/kg) and AQ gave parasite clearance of 55 and 95 % on day 5 of treatment. MD possesses antimalarial activity and is relatively safe.     

Impaired macrophage responses may contribute to exacerbation of blood-stage Plasmodium chabaudi chabaudi malaria in interleukin-12-deficient mice.

Aiming to clarify the role of endogenous interleukin-12 (IL-12) in protective immunity against blood stages of Plasmodium chabaudi chabaudi (AS), we evaluated the course of infection in IL-12p40 gene knockout (IL-12p40KO) and wild-type (WT) C57BL/6 mice, focusing (1) on the ability of T cells to develop adequate type 1 responses and (2) on the potentiality of macrophages to respond to parasites, interferon-gamma (IFN-gamma), or both. We observed that IL-12p40KO mice develop significantly higher parasitemias during the acute infection, although mice from both groups clear the parasites within a month and similarly eliminate a secondary challenge. Thus, fully protective immunity to P. c. chabaudi can be generated in the absence of IL-12. However, this cytokine may promote parasite control during the early phase of infection. The increased acute parasitemia of IL-12p40KO mice was associated with both impaired IFN-gamma and nitric oxide (NO) response by spleen cells. Because stimulation with recombinant IFN-gamma (rIFN-gamma) failed to improve the NO response in IL-12p40KO macrophages, we investigated whether these cells have an intrinsic defect. Analysis of peritoneal macrophages revealed that IL-12p40KO cells produce higher levels of transforming growth factor-beta1 (TGF-beta1) compared with WT cells and respond to infected erythrocytes or rIFN-gamma by releasing little NO. Moreover, IL-12p40KO macrophages had a severely impaired ability to internalize opsonized infected erythrocytes, suggesting that the low effector profile assumed by these cells may compromise antibody-mediated immunity. Taken together, our results support the idea that the absence of IL-12p40 not only affects IFN-gamma production but also has deep consequences in macrophage effector functions that may contribute to exacerbation of the early phase of P. c. chabaudi malaria.     

Altered response of CD4+ T cell subsets to Plasmodium chabaudi chabaudi in B cell-deficient mice

Mice depleted of B cells from birth by treatment with anti-_µantibodies can control but not clear an infection with the malariaparasite Plasmodium chabaudi chabaudi (AS). Splenic CD4⁺ T cellsfrom these mice were unable to mount a significant T_h2 responseto the parasite in vitro as shown by much lower precursor frequenciesof T_h cells for antibody production and of IL-4-producing cellscompared with the response of control-treated mice. CD4⁺ T cellsof the anti-_µ-treated mice which respond to antigens ofP. chabaudi chabaudi maintained a T_h1 phenotype throughout primaryinfection, in contrast to control mice in which a sequentialappearance of T_h1 and T_h2 responses was observed. These datashow that T_h1 responses in anti-_µ-treated mice are sufficientto control parasitemia but not to eliminate an infection. Thedata further suggest that depletion of B cells by treatmentwith anti-_µ; antibodies reduces the generation of theT_h2 subset during a primary response to P. chabaudi chabaudi.     

In vivo selection of populations of Plasmodium chabaudi chabaudi AS resistant to a monoclonal antibody that reacts with the precursor to the major merozoite surface antigen.

Mice bearing a hybridoma secreting a monoclonal antibody (MAb), MAb-3, which significantly delays the onset of a Plasmodium chabaudi chabaudi AS, but not P. chabaudi chabaudi CB, challenge parasitemia in a passive transfer assay and which is specific for the precursor to the major merozoite surface antigen (PMMSA) of P. chabaudi chabaudi AS, were challenged intravenously with 10(3) P. chabaudi chabaudi AS-parasitized erythrocytes. The resultant parasitemia was very similar to that in normal mice except that initially the parasitemia was sometimes slightly delayed. Parasites derived from cryopreserved stabilates isolated from MAb-3 hybridoma mice with an unmodified parasitemia, or with a delayed parasitemia, were found to have lost their susceptibility to MAb-3 in the passive transfer assay. A number of anti-PMMSA MAb were used to immunoprecipitate lysates of parasite populations isolated directly from hybridoma-bearing mice. In some instances and with certain of the MAb, immunoprecipitation patterns were modified, but other isolates were not detectably different when compared with unselected P. chabaudi chabaudi AS parasites. Using a panel of MAb reacting with the PMMSA of P. chabaudi chabaudi AS, immunoprecipitation patterns of parasites derived from cryopreserved stabilates isolated from hybridoma-bearing mice were determined at 2-h intervals through the appropriate part of the parasite maturation cycle. In these derived populations, resistance to MAb-3 was not associated with a change in the immunoprecipitation reaction with the MAb used. These results are discussed in the context of current knowledge of genotypic and phenotypic antigenic diversity of malaria parasites and other protozoa.     

Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi in mice

Important to malaria vaccine design is the phenomenon of "strain-specific" immunity. Using an accurate and sensitive assay of parasite genotype, real-time quantitative PCR, we have investigated protective immunity against mixed infections of genetically distinct cloned "strains" of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice. Four strains of P. c. chabaudi, AS, AJ, AQ, and CB, were studied. One round of blood infection and drug cure with a single strain resulted in a partial reduction in parasitemia, compared with levels for na?ve mice, in challenge infections with mixed inocula of the immunizing (homologous) strain and a heterologous strain. In all cases, the numbers of blood-stage parasites of each genotype were reduced to similar degrees. After a second, homologous round of infection and drug cure followed by challenge with homologous and heterologous strains, the parasitemias were reduced even further. In these circumstances, moreover, the homologous strain was reduced much faster than the heterologous strain in all of the combinations tested. That the immunity induced by a single infection did not show "strain specificity," while the immunity following a second, homologous infection did, suggests that the "strain-specific" component of protective immunity in malaria may be dependent upon immune memory. The results show that strong, protective immunity induced by and effective against malaria parasites from a single parasite species has a significant "strain-specific" component and that this immunity operates differentially against genetically distinct parasites within the same infection.     

Immunoglobulin E elevation in Plasmodium chabaudi malaria.

In order to investigate the mechanism of immunoglobulin E (IgE) elevation in malaria we studies mice infected with asexual blood stages of the rodent malaria parasite Plasmodium chabaudi chabaudi for total IgE and IgE antimalarial antibodies. Multiply infected mice had elevated levels of total as well as malaria-specific IgE in their sera. Sera taken from mice 3 weeks after one infection with P. chabaudi showed no IgE elevation, indicated that prolonged or repeated exposure to the parasite is necessary for the induction of an IgE response, which also is induced independently of previous or simultaneous infection with other pathogens such as helminths.     

Human recombinant tumor necrosis factor alpha protects susceptible A/J mice against lethal Plasmodium chabaudi AS infection.

The effect of intravenous treatment with human recombinant tumor necrosis factor alpha (rTNF-alpha) on infection of susceptible A/J and resistant C57BL/6 mice with Plasmodium chabaudi AS was examined. Treatment of A/J mice with 10(3) or 10(5) U of rTNF-alpha on days 0, 3, 5, 7, and 9 after intraperitoneal infection with 10(6) parasitized erythrocytes resulted in 80% survival and a significant decrease in the peak parasitemia level. Treatment of susceptible A/J hosts with 10(5) but not 10(3) U of rTNF-alpha resulted in increased survival but did not alter the peak parasitemia level following infection with 10(7) parasitized erythrocytes. Moreover, all surviving A/J mice completely eliminated the parasite by approximately 4 weeks and were fully protected against a secondary infection. Except at a dose of 5 x 10(5) U of rTNF-alpha, which resulted in 100% mortality of infected animals, rTNF-alpha did not alter the course or outcome of infection with P. chabaudi AS in resistant C57BL/6 mice.     

Lipoproteins and malaria. I. Immunogenicity of serum lipoproteins in mice infected with Plasmodium chabaudi

Anti-lipoprotein antibodies (anti-Lp Ab) have been investigated during the course of acute infection with P. chabaudi in Swiss mice using a radio-immunoprecipitation assay with (125)-iodine radiolabelled lipoproteins from normal or infected mice. Antibodies were detected 11 days after the beginning of infection; however, the highest lipoprotein precipitations were observed with purified (125)-I labelled lipoproteins from day-5 or day-7 infected mice. P. chabaudi infected mice were treated with chloroquine at various intervals after the beginning of infection and anti-Lp Ab were assayed on day 13. Anti-Lp Ab were not observed in mice treated before day 7 but were present in all mice treated after day 7. Anti-Lp Ab were not detected in mice infected with P. yoelii 17 X. Injection of purified lipoproteins from day-7 P. chabaudi infected mice (Lp day-7) to normal uninfected mice did not induce an antibody response to lipoproteins but anti-Lp Ab were observed when the same injection was performed in P. yoelii infected mice. Moreover, anti-Lp Ab were detected in uninfected mice injected concomitantly with Lp day-7 and hematin extracted from malarial pigment. Our results suggest that anti-Lp Ab observed in P. chabaudi infected mice are mainly against modified lipoproteins produced during infection and that the induction of the antibody response against lipoproteins requires an adjuvant effect such as the hematin which is released during infection.