Plasmodium berghei: influence on granulopoiesis and macrophage production in BALB/c mice

Granulocyte and macrophage progenitor cells forming colonies in vitro (GM-CFC) from bone marrow, spleen, and peripheral blood of BALB/c mice infected with Plasmodium berghei were cultured at various times postinfection in a viscous, 0.8% methylcellulose system. The numbers of GM-CFCs from bone marrow increased gradually during the first week of infection, reaching a maximum around the tenth day of the disease. Subsequently, a rise of GM-CFCs in cultures of nucleated cells from the peripheral blood was observed and, with some delay, in spleen cell cultures also, with a maximum around the end of the second week. After the tenth day of malaria infection a fall of colony frequency in bone marrow-derived cells took place, leading to subnormal values of GM-CFCs during the third week of infection. Subsequently, a decrease in the spleen cell cultures followed, but colony numbers did not fall to normal values. The general increase in GM-CFCs in the different organs was preceded by a rise in serum levels of colony-stimulating activity (CSA), attaining a maximum 1 week after P. berghei inoculation. During the following period the CSA levels fell and reached normal values around the seventeenth day of the disease. Chemotherapy with chloroquine started on the fifteenth day of infection, when GM-CFCs in the bone marrow have dropped to normal values, stopped their further decrease. In the spleen a gradual normalization took more than 2 weeks. A challenge infection evoked an elevation of GM-CFC numbers in the bone marrow and in the spleen during the first 10 days in only about 50% of immune mice. The reaction was immediate in some animals, but generally lower and of shorter duration than during primary infection. The results have indicated that a lethal P. berghei infection in mice caused a transient increase in production of CSA followed by a general recruitment of GM-CFCs in all hemopoietic organs.     

Induction of experimental autoimmune thyroiditis in mice with in vitro activated splenic T cells

Spleen cells from CBA/J or SJL mice sensitized with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) could be activated in vitro with MTg to transfer experimental autoimmune thyroiditis (EAT) to normal syngeneic recipients. EAT induced by these transferred cells was similar in incidence and severity to EAT induced by active immunization of mice with MTg and adjuvant and cells from EAT-resistant Balb/c mice could not be activated to induce EAT. The specific antigen MTg was required both for initial sensitization of the mice and for activation of spleen cells in vitro. The cells that were active in transferring EAT to mice were shown to be T cells. Removal of B cells from the cultured spleen cells had no effect on the ability of the cells to induce EAT.     

An effective diaryl derivative against Leishmania amazonensis and its influence on the parasite X macrophage interaction

The activity of several diarylheptanoid derivatives (curcuminoids) was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was 5-hydroxy-7- (4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)-1,4,6-heptatrien-3-one. This study was carried out to investigate the influence of this diaryl derivative on the infective promastigotes and Balb/c mice peritoneal macrophage interaction. The potential in vitro toxicity was also evaluated. Promastigotes pretreated for 24 hours with the compound had their infective capacity significantly decreased. When the infection of Balb/c macrophage by L. amazonensis promastigotes was already installed, addition of the drug resulted in a diminishing of the infection rate. It was demonstrated that the compound was not toxic to the host macrophage in a concentration equivalent to the LD50/24h from the previous in vitro experiment.     

Quantifying macrophage defects in type 1 diabetes

Macrophages from animals prone to autoimmune (type 1) diabetes differ from those of diabetes-resistant animals in processing and clearing apoptotic cells. Using in vitro time-course assays of the number of engulfed apoptotic cells observed within macrophages, we quantified these differences in non-obese diabetic (NOD) versus Balb/c mice. Simple models lead to several elementary parameter estimation techniques. We used these to compute approximate rates of macrophage engulfment and digestion of apoptotic cells from basic features of the data (such as initial rise-times, phagocytic index and percent phagocytosis). Combining these estimates with full fitting of a sequence of model variants to the data, we find that macrophages from normal (Balb/c) mice engulf apoptotic cells up to four times faster than macrophages from the diabetes-prone (NOD) mice. Further, Balb/c macrophages appear to undergo an activation step before achieving their high engulfment rate. In NOD macrophages, we did not see evidence for this activation step. Rates of digestion of engulfed apoptotic cells by macrophages are similar in both types. Since macrophage clearance is an important mechanism of disposal of self-antigen, these macrophage defects could potentially be a factor in predisposition to type 1 diabetes.     

Suppression of malaria-induced autoimmunity by immunization with cryoglobulins

Cryoglobulins obtained from malaria-infected (Plasmodium berghei berghei) Balb/c mice were administered intraperitoneally to naive Balb/c mice. Ten days or 9 months following cryoglobulin administration, the naive mice were infected with malaria. Comparison of sera from cryoglobulin-treated malaria-infected mice with sera from control infected mice revealed that pretreatment with cryoglobulins resulted in (1) reduced levels of circulating immune complexes; (2) reduced levels of autoantibodies reactive with nuclear and cytoplasmic antigens; and (3) suppressed development of cryoglobulinemia. Furthermore, the effect of cryoglobulins was long lasting, suggesting that recipient mice may have been actively immunized against autoantibody production.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

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.     

Scant parasitemia in BALB/c mice with congenital malaria infection

Balb/c mice were examined to determine whether or not they transmitted rodent malaria, Plasmodium berghei, to their fetuses. On the 15th day of pregnancy, mice were inoculated with approximately 3 x 10(6) P. berghei-infected erythrocytes by peritoneal injection. The blood from 27 adult females and 196 neonates was examined using a sensitive polymerase chain reaction (PCR) method with a detection level of approximately 1 parasite/microl blood. The average parasitemia of females at delivery was 8.1%, ranging from nondetectable to 37.1%. In 12 females, nested PCR established the presence of blood parasite DNA. Malaria parasites were microscopically confirmed in 2 of the 12 neonates. Maternal parasitemia at the time of delivery was not correlated with the incidence of vertical infection (6.1%), which was higher in this study than that found in previous studies. Although the combination of balb/c mice and P. berghei has not been used to examine vertical transmission of malaria, our report showed that this model may be used for this purpose.     

Cell-mediated immunity in rats injected with an antimalaria T-cell line

The aim was to develop a pure T-cell line which would enable the study of some aspects of the cellular immunity of malaria-injected rodents. For this purpose a long-term proliferative antimalaria T-cell line (AMTL) was established. The line was developed from splenocytes of rats recovered from a Plasmodium berghei infection. After adoptive transfer of the AMTL, some protection was demonstrated either by a lower mortality rate after challenge with the parasite or by decreased parasitemia in the T-cell injected versus control groups. Specific delayed-type hypersensitivity (DTH) was elicited by the AMTL: normal rats injected with the AMTL showed a DTH reaction to malaria antigens similar to the response observed in convalescent animals. Specificity was demonstrated by a marked response of the AMTL injected rats compared to rats injected with a control line (anti-purified protein derivative of tuberculin).     

Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57Bl/6 mouse

ABSTRACT: BACKGROUND: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. METHODS: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce cerebral malaria (CM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. RESULTS: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from CM. Nevertheless, infection with parasites of the ANKADeltapm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. CONCLUSIONS: Infection of pregnant C57BL/6 females with K173, NK65 and ANKADeltapm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.     

Dextran sulfate induced suppression of Plasmodium berghei parasitaemia

Effect of dextran sulfate (DS, Mr 500,000) on parasitaemia in mice (Balb/c) infected with erythrocytic stage of P. berghei was investigated. Intraperitoneal injection of DS caused marked suppression of patent parasitaemia and also enhanced the survival time of the infected animals.     

Heterogeneous and tissue-specific regulation of effector T cell responses by IFN-gamma during Plasmodium berghei ANKA infection

IFN-γ and T cells are both required for the development of experimental cerebral malaria during Plasmodium berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4(+) and CD8(+) T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN-γ(-/-) mice during P. berghei ANKA infection. The expansion of splenic CD4(+) and CD8(+) T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ, but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN-γ(-/-) mice; however, the migration to, and accumulation of, effector CD4(+) and CD8(+) T cells in the lung, liver, and brain was altered in IFN-γ(-/-) mice. Interestingly, activation and accumulation of T cells in various nonlymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN-γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended on active IFN-γ-dependent environmental signals--leading to T cell apoptosis--rather than upon intrinsic alterations in T cell programming. To our knowledge, this is the first study to fully investigate the role of IFN-γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.     

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.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

The influence of dietary fish-oil supplementation on cutaneous Leishmania amazonensis infection in mice

Dietary fish-oil (FO) supplementation has been shown to inhibit inflammation in various clinical disease states and to be beneficial in experimental models of inflammation and bacterial and plasmodial infection. In mice, FO increases macrophage production of tumour necrosis factor alpha (TNF). Production of TNF has been reported to be important in the resistance of mice against various Leishmania spp. We investigated whether dietary supplementation with FO protects susceptible Balb/c mice against infection with Leishmania amazonensis. No influence of the FO diet on the course of infection was observed, as evaluated by the increase in thickness of infected footpads over forty days. Lipopolysaccharide (LPS)-induced TNF production of peritoneal cells was however significantly increased in FO fed mice (P<0.01). When L. amazonensis was used as a stimulus, the in-vitro production of TNF by isolated peritoneal cells was minimal and did not differ between the various treatment groups. Addition of interferon gamma did not restore the effect of FO on TNF production capacity. We conclude that dietary supplementation with FO is of no benefit in Leishmaniasis in susceptible Balb/c mice, and that L. amazonensis is an insufficient trigger for TNF production in this model.     

Plasmodium berghei: immunosuppression of the cell-mediated immune response induced by nonviable antigenic preparations

In this work, plasmodial antigens were examined for their ability to suppress the cellular immune response during lethal Plasmodium berghei infection. Splenic enlargement and the number and function of white spleen cells were assessed after injection of normal mice with irradiated parasitized erythrocytes (IPE) or with parasitized erythrocytes (PE) membranes. Both IPE and PE membranes caused splenomegaly and an increase in the number of splenic white cells with concurrent alteration of the relative proportions of T cells and macrophages. The percentage of T lymphocytes was fractionally diminished, but there was a marked increase in Lyt 2.2 positive (suppressor and cytotoxic) T subsets and in the number of splenic macrophage precursors. The pathological enlargement of the spleen was induced by various plasma membrane-derived antigens containing both proteins and carbohydrates. Splenocytes of mice injected with liposomes containing deoxycholate-treated PE or PE fractions showed both diminished interleukin 2 production and a decreased response to mitogen. It appears that some of the changes in the cellular immune response during P. berghei infection are a consequence of the massive provision of a wide spectrum of antigens, capable of suppressing the immune response. Thus, it may be appropriate to evaluate the possible negative effect of parasite epitopes that are candidates for vaccine.     

Macrophage migration inhibitory factor: a downregulator of early T cell-dependent IFN-gamma responses in Plasmodium chabaudi adami (556 KA)-infected mice

Neutralization of macrophage migration inhibitory factor (MIF) increases anti-tumor cytotoxic T cell responses in vivo and IFN-γ responses in vitro, suggesting a plausible regulatory role for MIF in T cell activation. Considering that IFN-γ production by CD4(+) T cells is pivotal to resolve murine malaria and that secretion of MIF is induced by Plasmodium chabaudi adami parasites, we investigated the effect of MIF deficiency on the infection with this pathogen. Infections with P. c. adami 556 KA parasites were more efficiently controlled in MIF-neutralized and MIF-deficient (knockout [KO]) BALB/c mice. The reduction in parasitemia was associated with reduced production of IL-4 by non-T/non-B cells throughout patent infection. At day 4 postinfection, higher numbers of activated CD4(+) cells were measured in MIF KO mice, which secreted more IFN-γ, less IL-4, and less IL-10 than did CD4(+) T cells from wild-type mice. Enhanced IFN-γ and decreased IL-4 responses also were measured in MIF KO CD4(+) T cells stimulated with or without IL-12 and anti-IL-4 blocking Ab to induce Th1 polarization. However, MIF KO CD4(+) T cells efficiently acquired a Th2 phenotype when stimulated in the presence of IL-4 and anti-IL-12 Ab, indicating normal responsiveness to IL-4/STAT6 signaling. These results suggest that by promoting IL-4 responses in cells other than T/B cells during early P. c. adami infection, MIF decreases IFN-γ secretion in CD4(+) T cells and, additionally, has the intrinsic ability to render CD4(+) T cells less capable of acquiring a robust Th1 phenotype when stimulated in the presence of IL-12.     

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.     

Characterization of the immuno-regulatory response to the tapeworm Hymenolepis diminuta in the non-permissive mouse host

Hymenolepis diminuta is spontaneously expelled from mice; concomitant with worm expulsion was protection against colitis induced by dinitrobenzene sulphonic acid (DNBS). Here we examined the immune response mobilized by Balb/c and C57Bl/6 male mice in response to H. diminuta and assessed the requirement for CD4+ cells (predominantly T cells) in worm expulsion and the anti-colitic effect. Wild-type (CD4+) or CD4 knock-out (CD4-/-) mice received five H. diminuta cysticercoids and segments of jejunum and mesenteric lymph nodes (MLNs), or spleen, were excised 5, 8 and 1l days later for mRNA analysis and cytokine production, respectively. In separate experiments uninfected and infected mice received DNBS by intra-rectal infusion and indices of inflammation were assessed 3 days later (i.e. 11 days p.i.). Infection of Balb/c mice resulted in a time-dependent increase in intestinal mRNA for Foxp3, a marker of natural regulatory T cells, and markers of alternatively activated macrophages (arginase-1, FIZZ1), while concanavalin-A activation of MLN cells revealed a significant increase in T helper 2 (TH2) type cytokines: IL-4, -5, -9, -10, -13. MLN cells showed a reduced ability to induce Foxp3 expression upon stimulation. CD4-/- mice did not display this response to infection, but surprisingly did expel H. diminuta. Moreover, DNBS-induced colitis in CD4-/- mice (wasting, tissue damage, elevated myeloperoxidase) was not reduced by H. diminuta infection, whereas time-matched infected CD4+ C57Bl/6 mice had significantly less DNBS-induced inflammation. In conclusion: (i) in addition to stereotypical TH2 events, H. diminuta-infected Balb/c mice develop a local immuno-regulatory response; and (ii) CD4+ cells are not essential for H. diminuta expulsion from mice but are critical in mediating the anti-colitic effect that accompanies infection in this model.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.