Antibodies to Voltage-Gated Calcium Channels in Children with Falciparum Malaria

    Neurosciences and Molecular Parasitology Groups, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford
    Neurosciences Department, Institute of Child Health, London, United Kingdom
    Centre for Geographic MedicineCoast, Kenya Medical Research Institute, Kilifi, Kenya

    Falciparum malaria can affect the central nervous system (CNS), causing neurological dysfunction and sequelae. The pathophysiology of these complications is currently very poorly understood. Production of autoantibodies has frequently been reported as a consequence of infection with Plasmodium falciparum. However, at present, the presence of antibodies to components of the CNS during malaria infection has not been reported. We have sought to identify such antibodies, define their specificity, and determine whether they are involved in the development of neurological complications of falciparum malaria. Here, we show that, in a cohort of Kenyan children, levels of antibodies to the voltage-gated calcium channels, but not to other ion channels, increased with the severity of malaria infection.

    Malaria is a major cause of morbidity and mortality in children living in sub-Saharan Africa. The clinical consequences of infection with Plasmodium falciparum range from asymptomatic infection, to mild illness, to severe life-threatening disease. Many children present with symptoms of fever, headache, and drowsiness, which will either spontaneously resolve or respond to treatment. The main neurological manifestations are seizures and impaired consciousness; the clinical syndrome of cerebral malaria (CM) is the most severe [1]. Unusually, the majority of African children with CM who survive recover without obvious neurological deficits. However, 11% of these children are discharged with neurological sequelae, such as ataxia, hemiparesis, or visual impairment [1]. Some children develop psychosis and hallucinations during or after the infection. There is also an increased risk of developing epilepsy in these children [2]. The pathogenesis of the neurological complications that follow falciparum malaria is unclear, since the parasites do not penetrate the brain parenchyma.

    Autoantibodies to a restricted set of self-proteins are a feature of some central nervous system (CNS) disorders [3]. Antibodies to the glutamate receptors, phospholipids, cardiolipin, and glutamic acid decarboxylase (GAD) are associated with different forms of epilepsy [4], whereas antibodies to voltage-gated potassium channels (VGKCs) and voltage-gated calcium channels (VGCCs) have been associated with certain forms of limbic encephalitis [5] and cerebellar ataxia [3]. Since autoantibodies have frequently been reported to be associated with malaria infection (although not with specificity for components of the CNS), we were interested to determine whether autoantibodies with CNS specificity were produced as a result of malaria infection and whether they were associated with neurological manifestations. In the present study, we looked for the presence of autoantibodies to various components of the nervous system, both peripheral and central, in the blood of children with uncomplicated and severe falciparum malaria and compared the levels with those measured in children in the local community.

    PATIENTS AND METHODS

    Patients.

    Plasma samples were collected from Kenyan children who were admitted to Kilifi District Hospital with a primary diagnosis of severe malaria, from those with mild malaria who attended the hospital outpatient department, and from a set of age-matched control subjects within the community. Malaria case subjects were defined as those children with P. falciparum parasitemia (asexual P. falciparum parasites detected on a peripheral film stained with 10% Giemsa)(1) cases of CM were defined in children who could not localize a painful stimulus and for whom other causes of encephalopathy had been excluded [6], (2) cases of malaria plus seizures (M+S) were defined in children with a history of 2 convulsions during the 24-h period before admission, and (3) cases of mild malaria (MM) were defined in children attending the outpatient clinic in Kilifi district hospital who received a primary diagnosis of malaria but had none of the above complications and were not admitted to the hospital. In addition, antibodies were measured in British children with bacterial meningitis (n = 10). All experiments involving humans followed published guidelines of conduct of clinical research [7] and were performed after informed consent of the children's parents or guardians had been obtained. The present study was approved by the Kenya National Ethical Committee.

    Methods.

    Plasma samples were assayed for antibodies to P-/Q- and N-type VGCCs, VGKCs, and muscle nicotinic acetylcholine receptors (nAChRs), as described elsewhere [8, 9]. In brief, human extracts of brain or muscle (510 fmol) were labelled with [125I]-neurotoxins specific for the channels to be labelled ([125I]--conotoxin MVIIC, [125I]--conotoxin GVIA, [125I]-dendrotoxin, and [125I]--bungarotoxin, which label P-/Q-type VGCCs, N-type VGCCs, VGKCs, and muscle nAChRs, respectively). The labelled extract was incubated overnight in patients' serum samples (510 L), and the complex was precipitated by the addition of goat antihuman IgG serum. The samples were centrifuged, washed, and counted. Antibodies to the -aminobutyric acid synthesizing enzyme, GAD, were measured by use of a radioimmunoassay kit developed by RSR. To account for the higher levels of immunoglobulin found in African children, each assay was initially standardized with plasma (n = 10) from healthy age-matched community control subjects. These samples were run in all subsequent assays, and their mean value was subtracted from that of the test samples. The results are expressed as the mean of 3 separate determinations. All assays were performed by investigators who were blinded to the clinical status of the children.

    RESULTS

    Of the 49 children with CM, 35 recovered (mean ± SD antibody titer, 81.5 ± 121 pmol/L), 8 had residual neurological sequelae (mean ± SD antibody titer, 148 ± 133 pmol/L), and 6 died (mean ± SD antibody titer, -29 ± 46 pmol/L). None of the 6 children who died had a positive antibody titer. There was no significant difference between the antibody titers of children with CM who either recovered, had residual sequelae, or died.

    A number of different subtypes of VGCCs have been described on the basis of their biophysical and pharmacological properties. We have assessed the samples for P-/Q- and N-type VGCCs. In the present study, all patients with N-type VGCC antibodies also had P-/Q-type VGCC antibodies, and there was a strong correlation between P-/Q-type and N-type VGCC antibody titers (figure 3).

    DISCUSSION

    Antibodies to VGCCs, but not to other ion channels, are detected, at increased frequency and concentration, in children with malaria. This is not due to a general increase in circulating antibodies or general polyclonal activation; this trend is not observed in any of the other antibody assays. There is a trend for increasing levels of antibody, first with infection per se and subsequently with disease severity. This suggests that the detectable levels of these antibodies may be a marker of current infection and that higher levels of antibody are associated with disease severity. Furthermore, the levels of anti-VGCC antibody were significantly higher in children with malaria and a history of seizures (CM and M+S groups) than in those without seizures (MM). Although the antibody titers were highest in the children with CM, within that group, none of the children who died had detectable levels of anti-VGCC antibodies; however, the numbers of children in each group were too small to be significant.

    Autoantibodies to VGCCs are seen in a limited range of autoimmune disorders, including Lambert-Eaton myasthenic syndrome (LEMS) and paraneoplastic cerebellar ataxia [10], but, at present, have not been reported in association with infection. In LEMS, the antibodies are highly specific to P-/Q-type VGCCs: >90% of patients have positive titers, whereas only 30% have antiN-type VGCC antibodies [9]. It is the P-/Q-type VGCC that is considered to be important in modulation of neurotransmission in the CNS. AntiP-/Q-type, but not antiN-type, VGCC antibodies have been shown to produce pathogenic effects [11]. In contrast, the present study has shown that anti-VGCC antibodies recognize P-/Q-type and N-type VGCCs almost equally, and this correlation may indicate cross-reactivity with a common epitope, either in the channel itself or in a closely associated protein. Alternatively, the VGCC is known to exist in a large complex of proteins, including other molecules involved in neurotransmitter release, and, so, the possibility occurs that the antibodies are in fact recognizing other molecules, such as syntaxin and synaptotagmin, that coprecipitate with the labelled VGCCs [12].

    The present study has shown a weak correlation between parasitemia and levels of antibody. Peripheral parasitemia is not a good measure of total parasite load in malaria, because of the stage-specific sequestration of infected cells within the deep vasculature. This weak but significant correlation may therefore reflect a much tighter association between parasite load and levels of antibody. The lack of a clear correlation between the antibody titer and the severity of clinical symptomsfor example, some children with CM have low antibody titershas also been seen in a number of different established autoimmune disorders. For example, in LEMS, the absolute antibody titer does not correlate with clinical severity in the population as a whole, although there is often an inverse relationship between antibody titer and disease severity in an individual.

    Several possible mechanisms may be involved in the generation of these seemingly specific antibodies. Since they were not detected in patients with bacterial meningitis, they are unlikely to be a generalized feature of CNS infection. P. falciparum has been reported to induce polyclonal B cell activation, but the lack of antibodies to other channels makes this an unlikely explanation. Alternatively, it is possible that these antibodies are generated to cross-reactive P. falciparum epitopes. Although we cannot eliminate the possibility of cross-reactive conformational epitopes, exhaustive searching of the entire P. falciparum genome sequence (available at: http://www.PlasmoDB.org) revealed no primary sequence homology to either P-/Q- or N-type channels. Finally, it may be that the impairment of the blood-brain barrier in African children with cerebral malaria [13] and increased levels of IgG [14, 15] may allow an antibody response to normally inaccessible determinants to which the host has not developed tolerance. The seizures themselves may also result in cell damage and subsequent generation of antibodies. However, the absence of antibodies to other CNS components, such as VGKCs and GAD, does not support this. The detection of autoantibodies in the cerebrospinal fluid (CSF) is problematic because of the sensitivity of the assay systems involved. We have assayed CSF from 10 patients with elevated anti-VGCC titers. A titer of 28 pmol/L, which was significantly different from the mean ± SD antibody titer from CSF from African children without serum antibodies (-14 ± 10 pmol/L), was detected in one patient with a serum antibody titer of 240 pmol/L. In a recent study [10] of paired serum/CSF samples from patients with anti-VGCC antibodies and paraneoplastic cerebellar ataxia, anti-VGCC antibodies were detected for 7 of 15 patients; however, the levels of antibody in the CSF (range, 20400 pmol/L) were 1/100 those in the serum (range, 10030,000 pmol/L).

    We hypothesize that these autoantibodies, induced by P. falciparum, may be involved in the generation of some of the neurological complications of acute falciparum malaria and/or the subsequent development of complications, such as cerebellar ataxia [16] and epilepsy [2]. Further studies are required to investigate the pathogenicity of these antibodies and to study the relationship between their generation and the subsequent development of epilepsy.

    Acknowledgments

    We would like to thank Nigel Klein (Great Ormond Street Hospital, London) for providing the plasma samples from patients with bacterial meningitis.

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