Charlotte Behr

Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria

BACKGROUND Severe anaemia is a major complication of malaria but little is known about its pathogenesis. Experimental models have implicated tumour necrosis factor (TNF) in induction of bone-marrow suppression and eythrophagocytosis. Conversely, interleukin 10 (IL-10), which mediates feed-back regulation of TNF, stimulates bone-marrow function in vitro and counteracts anaemia in mice. We investigated the associations of these cytokines with malarial anaemia. METHODS We enrolled 175 African children with malaria into two studies in 1995 and 1996. In the first study, children were classified as having severe anaemia (n=10), uncomplicated malaria (n=26), or cerebral anaemia (n=41). In the second study, patients were classified as having cerebral malaria (n=33) or being fully conscious (n=65), and the two groups were subdivided by measured haemoglobin as normal (>110 g/L), moderate anaemia (60-90 g/L), and severe anaemia (<50 g/L). IL-10 and TNF concentrations were measured by ELISA in plasma samples from all patients. FINDINGS IL-10 concentrations were significantly lower in patients with severe anaemia than in all other groups. In 1995, geometric mean plasma IL-10 in patients with severe anaemia was 270 pg/mL (95% CI 152-482) compared with 725 pg/mL (465-1129) in uncomplicated malaria and 966 pg/mL (612-1526) in cerebral malaria (p<0.03). In 1996, fully conscious patients with severe anaemia also had significantly lower IL-10 concentrations than all other groups, including cerebral-malaria patients with severe anaemia and all patients with moderate anaemia (p<0.001). In both studies, TNF concentrations were significantly higher in cerebral malaria than in fully conscious patients (p<0.01). By contrast, the ratio of TNF to IL-10 was significantly higher in fully conscious patients with severe anaemia than in all other groups (p<0.001). INTERPRETATION Our findings identify severe malarial anaemia as a distinct disorder in which insufficient IL-10 response to high TNF concentrations may have a central role.

Increased Levels of Inflammatory Mediators in Children with Severe Plasmodium falciparum Malaria with Respiratory Distress

BACKGROUND Respiratory distress (RD), a symptom of underlying metabolic acidosis, has been identified as a major risk factor for mortality in children with severe malaria in Africa, yet the molecular mediators involved in the pathogenesis of RD have not been identified. METHODS We studied circulating levels of mediators of inflammation--including the cytokines tumor necrosis factor (TNF)- alpha and interleukin (IL)-10; the chemokines macrophage inflammatory protein (MIP)-1 alpha , MIP-1 beta , and IL-8; and the immune activation marker neopterin--in children with RD, severe malarial anemia (SMA), cerebral malaria (CM), and uncomplicated malaria (UM). RESULTS Children with RD had significantly higher plasma levels of TNF- alpha , IL-10, and neopterin and a significantly higher TNF- alpha : IL-10 ratio than those without RD. In addition, the results demonstrated that, relative to UM, CM was associated with increased levels of TNF- alpha and decreased levels of MIP-1 alpha , whereas SMA was associated with decreased levels of IL-10. Circulating levels of neopterin were inversely correlated with hemoglobin, whereas levels of MIP-1 beta were positively correlated with parasitemia. CONCLUSIONS We conclude that distinct clinical presentations of severe malaria are associated with specific patterns of inflammatory mediators. In particular, we show, to our knowledge for the first time, that patients with malaria and RD have a strong and unbalanced proinflammatory response that may be involved in the pathogenesis of the underlying metabolic acidosis.

Perturbation and proinflammatory type activation of Vδ1+ γδ T cells in African children with Plasmodium falciparum malaria

ABSTRACT γδ T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the γδ T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of γδ T cells are transiently increased following treatment of Plasmodium falciparum malaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the γδ T cells involved in this perturbation expressed Vδ1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the Vδ1+ cell population at the peak of their increase showed that all expressed Vγ chains were used, and CDR3 length polymorphism indicated that the expanded Vδ1 population was highly polyclonal. A very high proportion of the Vδ1+ T cells produced gamma interferon, while fewer Vδ1+ cells than the average proportion of all CD3+ cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-γδ+cells in general or Vδ1+ cells in particular. Taken together, our data point to an immunoregulatory role of the expanded Vδ1+ T-cell population in this group of semi-immuneP. falciparum malaria patients.

Cytokine production and apoptosis among T cells from patients under treatment for Plasmodium falciparum malaria.

Available evidence suggests that Plasmodium falciparum malaria causes activation and reallocation of T cells, and that these in vivo primed cells re‐emerge into the periphery following drug therapy. Here we have examined the cytokine production capacity and susceptibility to programmed cell death of peripheral T cells during and after the period of antimalarial treatment. A high proportion of peripheral CD3+ cells had an activated phenotype at and shortly after time of admission (day 0) and initiation of therapy. This activation peaked around day 2, and at this time‐point peripheral T cells from the patients could be induced to produce cytokines at conditions of limited cytokine response in cells from healthy control donors. Activated CD8hi and TCR‐γδ+ cells were the primary IFN‐γ producers, whereas CD4+ cells constituted an important source of TNF‐α. The proportion of apoptotic T cells was elevated at admission and peaked 2 days later, while susceptibility to activation‐induced cell death in vitro remained increased for at least 1 week after admission. Taken together, the data are consistent with the concept of malaria‐induced reallocation of activated T cells to sites of inflammation, followed by their release back into the peripheral blood where they undergo apoptotic death to re‐establish immunological homeostasis as inflammation subsides. However, the high proportion of pre‐apoptotic cells from the time of admission suggests that apoptosis also contributes to the low frequency and number of T cells in the peripheral circulation during active disease.

Is there a role for γδ T cells in malaria

Abstract Peripheral blood lymphocytes of the V γ 9 + family of γδ T cells proliferate vigorously in response to Plasmodium falciparum . In this brief article, Jean Langhorne and colleagues discuss this response and assess the possible role of γδ T cells in the pathogenesis of malaria.

Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children

Severe malarial anaemia (SA) is a major complication of malaria and an important cause of child mortality and morbidity. However, the pathogenesis behind SA is poorly understood. Nitric oxide (NO) is known to play a protective role against clinical malaria but is also suggested to have a pathogenic role in cerebral malaria (CM). Erythrophagocytosis by splenic macrophages has been implicated in the pathogenesis of SA. In this study, plasma levels of NO, neopterin, haptoglobin and C-reactive protein (CRP) were measured in paediatric patients with CM, n=77, SA (n=28) and uncomplicated malaria (UM n=53). Haptoglobin levels were significantly lower in SA (median (interquartile range) 25 (17-59) mg/l) than in both CM and UM (40 (24-80) mg/l and 110 (60-160) mg/l, respectively, P<0.001). In contrast, NO levels were higher in SA (38 (28-51) micromol/l) than in CM and UM (21 (15-32) micromol/l and 10.3 (5.6-17) micromol/l, respectively, P<0.001). A significant negative correlation between haptoglobin and NO was seen in the SA group. No such correlation was observed within the UM or CM groups. No significant differences in neopterin levels were observed between any of the three groups, neither was there any correlation between parasitaemias and neopterin levels. The low haptoglobin and high levels of NO in this SA group may contribute to haemolysis. Taken together our results support the hypothesis that immune-mediated erythrocyte destruction is involved in the pathogenesis of malarial anaemia.

Plasmodium falciparumin the squirrel monkey (Saimiri sciureus): infection of non-splenectomised animals as a model for exploring clinical manifestations of malaria

Plasmodium falciparum infection in humans leads to a variety of symptoms ranging from an influenza-like syndrome to life-threatening complications. Animal models are useful tools for the detailed analysis of the interaction between both parasite and host factors leading to these various clinical manifestations. In this review, examining the different clinical, parasitological and haematological parameters associated with P. falciparum infection in spleen-intact monkeys, we propose this model as a good alternative for exploring some aspects of the host-parasite relationship in malaria.

Antitumor Activity of γδ T Cells Reactive against Cytomegalovirus-Infected Cells in a Mouse Xenograft Tumor Model

gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.

Insights into deregulated TNF and IL-10 production in malaria: implications for understanding severe malarial anaemia.

BackgroundSevere malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients.MethodsThe IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined.ResultsMonocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001).ConclusionsThese data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.