Levels of cytokines in plasma during Plasmodium falciparum malaria attacks.

The variation of levels of tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, gamma interferon, neopterin, and interleukin-2 receptors in plasma were monitored in 16 patients presenting with an acute Plasmodium falciparum malaria attack. Relations among cytokine levels and between cytokine levels and hematological and parasitological data were assessed.     

Low interleukin-12 activity in severe Plasmodium falciparum malaria

We compared interleukin-12 (IL-12) and other cytokine activities during and after an acute clinical episode in a matched-pair case-control study of young African children who presented with either mild or severe Plasmodium falciparum malaria. The acute-phase, pretreatment plasma IL-12 and alpha interferon (IFN-alpha) levels, as well as the acute-phase mitogen-stimulated whole-blood production capacity of IL-12, were significantly lower in children with severe rather than mild malaria. IL-12 levels, in addition, showed strong inverse correlations both with parasitemia and with the numbers of circulating malaria pigment-containing neutrophils. Acute-phase plasma tumor necrosis factor (TNF) and IL-10 levels were significantly higher in those with severe malaria, and the concentrations of both of these cytokines were positively correlated both with parasitemia and with the numbers of pigment-containing phagocytes in the blood. Children with severe anemia had the highest levels of TNF in plasma. In all the children, the levels in plasma and production capacities of all cytokines normalized when they were healthy and parasite free. The results indicate that severe but not mild P. falciparum malaria in young, nonimmune African children is characterized by down-regulated IL-12 activity, contrasting markedly with the up-regulation of both TNF and IL-10 in the same children. A combination of disturbed phagocyte functions resulting from hemozoin consumption, along with reduced IFN-gamma responses, may contribute to these differential effects.     

Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.     

Anemia and interleukin-10, tumor necrosis factor alpha, and erythropoietin levels among children with acute, uncomplicated Plasmodium falciparum malaria.

Anemia is an important complication of malaria, and its pathogenesis is not well understood. To gain insight into potential age-related relationships between tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), erythropoietin, and anemia during acute malaria, 273 children of ages 12 to 120 months presenting with acute, uncomplicated malaria in Kampala, Uganda, were monitored at enrollment and 3 and 7 days later. Younger children had higher geometric mean erythropoietin, TNF-alpha, and alpha(1)-acid glycoprotein (AGP) concentrations than older children. Univariate regression analysis revealed that age, log(10) erythropoietin levels, IL-10/TNF-alpha ratio, and AGP levels were each significantly associated with hemoglobin levels at baseline. Hemoglobin concentrations were inversely correlated with the log(10) erythropoietin level at all three visits. For the older age groups, higher levels of TNF-alpha were significantly associated with higher IL-10 levels at all three visits, but this relationship was significant only at baseline for younger children. These data suggest that younger children do not maintain IL-10 production in response to the inflammatory process, and this mechanism may contribute to the more severe anemia found in younger children. Acute malaria is an illness whose incidence and severity are largely age dependent. Further studies are needed to understand the relationships between age-related immune responses to malaria and their role in the pathogenesis of malarial anemia.     

Detection of anti-neutrophil cytoplasmic antibodies after acute Plasmodium falciparum malaria.

Four of 30 patients with Plasmodium falciparum infection in Bangkok, Thailand, were positive for anti-neutrophil cytoplasmic antibodies by indirect immunofluorescence 1 month after antimalarial therapy. No myeloperoxidase, proteinase 3, lactoferrin, or elastase reactivity was found. Since no evidence of vasculitis was seen in these patients, anti-neutrophil cytoplasmic antibody production in malaria-infected susceptible patients probably represents a secondary response, indicating neutrophil activation.     

Elevated levels of soluble CD14 in serum of patients with acute Plasmodium falciparum malaria

Serum sCD14, tumour necrosis factor-alpha (TNF-α), IL-6, and endotoxin were analysed in 45 patients with complicated malaria, in 14 patients with Gram-negative septicaemia and in 24 healthy subjects by ELISA. Malaria patients with renal failure (n = 16) had higher levels than patients without renal failure (n = 29) (8116+1440 μg/lversus 9453+1017 μg/lP<0.05) and both had higher levels than patients with septicaemia (6155+1635μg/l) and normal subjects (2776+747 μg/l). A significant correlation between sCD14 and IL-6 (r = 0.756) and TNF (r = 0.822) existed. However, no relation between sCD14 and serum endotoxin or indices of clinical disease severity (parasitaemia, fever, parasite or fever clearance time) was seen. Although the role of sCD14 in malaria remains to be determined, elevated levels may participate in the inflammatory response in complicated malaria.     

Increased gamma delta T cells in acute Plasmodium falciparum malaria

The T cell receptor of gamma delta is normally expressed on a small percentage of peripheral lymphocytes. Although the role of gamma delta T cells in the physiologic immune response is still unknown, there is accumulating evidence that gamma delta T cells may participate in the immune response to mycobacterial and other infectious organisms. In this study, we have quantitated the number of circulating gamma delta T cells during acute Plasmodium falciparum malaria. The results indicate that gamma delta T cells are elevated during the acute infection and remain elevated for at least 4 weeks during convalescence. T cells may participate in the immune response against P. falciparum by functioning as non-MHC restricted cytotoxic cells against intraerythrocytic parasites. Alternatively, lymphokines may be produced on antigen stimulation which may have antiparasitic activity.     

New B cell epitopes in the Plasmodium falciparum malaria circumsporozoite protein.

Most antibodies directed against the Plasmodium falciparum circumsporozoite (CS) protein react with its central domain, which contains about 40 repeats of the tetrapeptide Asn-Ala-Asn-Pro (NANP). To search for new epitopes in the non-repetitive part of the CS protein, we expressed the non-repetitive regions of the protein in E. coli as fusion proteins with mouse dihydrofolate reductase linked to six adjacent histidine residues. These fusion proteins were obtained at greater than 70% purity by a single Ni-chelate affinity chromatography step. Of the new epitopes defined in the C-terminal portion of the CS protein, three are located in a stretch of 65 amino acids immediately C-terminal of the protein's central repetitive domain. Pooled sera from inhabitants of a malaria-endemic area reacted with epitopes in this region of the molecule, and four mouse monoclonal antibodies to this region also reacted with the native CS protein on sporozoites. Two of the monoclonal antibodies reacted with a peptide PNDPNRNVD derived from a conserved region of the CS protein. The other two antibodies showed different reactivities to sporozoites of the NF54 and Ro59 parasite isolates. One, which reacted with a peptide ENANANNAV, recognized Ro59 but not NF54 sporozoites, while the other reacted with a small percentage of NF54 but not Ro59 sporozoites. Antibodies which react with non-repetitive regions of the CS protein could contribute to maintaining its genetic variability.     

T cell responses in acute falciparum malaria

T lymphocyte responses to malaria-specific antigens during acute falciparum malaria were studied to determine host-parasite interaction and its relation to the manifestations of the disease. The results indicate that while there is antigen-specific immunodepression, markedly elevated levels of soluble factors such as IL2 receptor, CD8 antigen and IFN-gamma suggest that there is intense concurrent cellular activation which however does not seem to be effective in controlling the infection. It is proposed that the cellular activation is to a large extent non-specific and polyclonal, and leads to the exaggerated production of cytokines and eventually immunopathology. Various mechanisms of immunodepression are discussed.     

Increased levels of soluble Fas ligand in serum in Plasmodium falciparum malaria

Levels of soluble Fas ligand (sFasL) in serum were elevated in patients with Plasmodium falciparum malaria and showed a significant decline during disease course. sFasL levels that were elevated before antimalarial treatment began correlated significantly with depressed total lymphocyte and T-cell counts. These data suggest that Fas-induced apoptosis might play a role in malaria-associated lymphopenia.     

Measurement of acute phase proteins for assessing severity of Plasmodium falciparum malaria.

Seventeen adult patients with acute Plasmodium falciparum malaria, admitted to the Hospital for Tropical Diseases, were studied. Serial measurements of the serum concentration of C-reactive protein, serum amyloid A protein, and percentage parasitaemia were determined, together with initial measurement of serum electrolytes, liver function, haemoglobin, white cell and platelet counts. Initial C-reactive protein and serum amyloid A concentrations were increased (C-reactive protein mean 49.0 mg/l serum amyloid A 28 mg/l) falling towards the normal range by the seventh day of treatment. There was a significant correlation between the pretreatment parasite count and clinical and laboratory markers of inflammation. C-reactive protein and serum amyloid A concentrations correlated inversely with the serum sodium. These results indicate that measurement of acute phase reactants such as C-reactive protein and serum amyloid A may prove valuable in assessing the severity of P falciparum malaria, and in following the response to antimalarial treatment.     

Plasmodium falciparum malaria vaccines in development

The development and implementation of a malaria vaccine would constitute a major breakthrough for global health. Recently, numerous new candidates have entered clinical testing, following strategies that are as diverse as the malaria cycle is complex. While promising results have been obtained, some candidate vaccines have not fulfilled expectations. The challenges are not merely scientific; further progresses will require the development of competent investigator networks, partnerships between academics, industry and funding agencies, and continuous political commitment. In this review, we present the developmental status of all malaria vaccine candidates that are currently in human clinical testing against Plasmodium falciparum, as well as selected malaria vaccine candidates at preclinical development stage, and discuss the main challenges facing the field of malaria vaccine development.     

Anti-phospholipid antibodies in patients with Plasmodium falciparum malaria.

Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease.     

Evolution of the levels of soluble interleukin-2 receptors during Plasmodium falciparum and P. vivax malaria.

Increased levels of soluble interleukin-2 receptors (IL-2R) in serum were observed in both Plasmodium falciparum- and P. vivax-infected individuals compared with nonparasitemic subjects. Clinical symptoms of P. falciparum malaria were associated with higher levels of soluble IL-2R. Temporal evolution in serum of IL-2R during the course of a malaria attack mimicked the kinetics of soluble IL-2R under experimental conditions.     

Bacterial infections and early Plasmodium falciparum malaria

Two cases of Plasmodium falciparum malaria associated with bacterial infections--streptococcus A septicemia and Legionnaires' disease--are described. The association of these two infections is probably not incidental, and the hypothesis of Plasmodium falciparum induced immuno-deficiency is discussed.     

Cellular immune responses to Plasmodium falciparum antigens in Gambian children during and after an acute attack of falciparum malaria.

Peripheral blood mononuclear cells from 63 Gambian children with acute Plasmodium falciparum malaria were examined for lymphoproliferation and interferon-gamma (IFN) production in response to stimulation by mitogens, malaria antigens and other soluble antigens. Mitogen or Candida-induced proliferation was not depressed during acute infection but was enhanced 2 to 4 weeks after treatment. Responses to partially purified soluble P. falciparum antigens were minimal or absent in all children in the acute phase but approximately 50% of the children responded by proliferation or IFN-gamma production during the 2 to 8 week convalescent period. These proliferative responses were severely depressed in the presence of the patient's own serum. Nine children with significant convalescent phase proliferative responses were re-examined several months after acute infection. Of these, four remained responsive for at least 8 months in the probable absence of reinfection.     

Malaria mimicry with tumor necrosis factor. Contrasts between species of murine malaria and Plasmodium falciparum.

Because Plasmodium berghei ANKA induces cerebral malaria and P. vinckei does not, the former has often been studied as a model for human falciparum malaria. It lacks, however, many of the systemic changes seen in the human disease. Because both of these murine models and the human disease have now been defined in terms of excess tumor necrosis factor (TNF) production, the authors have more closely examined the two murine models in this light to see which provides the better overall model for falciparum malaria. Administering TNF to malaria-infected mice did not cause cerebral symptoms nor breakdown of the blood-brain barrier, which is the hallmark of P. berghei ANKA cerebral malaria and is generally absent in human cerebral malaria. Tumor necrosis factor did, however, induce hypoglycemia and liver injury, pathology that is seen in terminal P. vinckei and falciparum malaria, but is absent in terminal P. berghei ANKA malaria. Plasma TNF and interleukin-6 (IL-6) also were found to be consistently higher in infections caused by P. vinckei than in those caused by P. berghei ANKA. The pathology of P. vinckei malaria is thus consistent with raised systemic levels of TNF and other cytokines, as is falciparum malaria. The authors therefore conclude that P. vinckei malaria, although lacking a cerebral component, is the better model for the human disease.