Attempts to explain the pathogenesis of severe falciparum malaria have, in recent years, been dominated by two approaches now generally considered complementary: poor tissue oxygen supply due to the obstructive effects of microvascular sequestration  and the local and systemic effects of the excessive release of the cytokines that regulate cell-mediated immunity . In this study we investigated these interactions further, through immunohistochemical staining for MIF and iNOS in brain and other tissues from paediatric autopsies in Malawi. iNOS protein expression in the brain in fatal malaria has been reported in one previous study, of adults in south-east Asia . Sampling the cerebrum only, these authors reported immunohistochemical staining for iNOS in a range of cell types, most strongly in endothelial cells, in 13 cases of adult cerebral malaria. Bacterial septicemia cases were not studied. Judging from the universal presence of microhemorrhages reported in all brains examined, these adult brains correspond closest to the Category C subgroup of our paediatric cerebral malaria cases.
Tissues from systemic human disease have not previously been examined for MIF. The normal presence of MIF protein in bronchial epithelial cells, epidermis and subcutaneous glandular structures, as we have found in these human tissues, has previously been reported, by this same technique, in the rat . It has not previously been reported in skeletal muscle. A function of MIF possibly relevant to the pathology of severe malaria and sepsis, and consistent with its presence in skeletal muscle, is its ability to increase synthesis of fructose 2,6-bisphosphate (F2,6BP), a positive allosteric regulator of glycolysis . A neutralising anti-MIF mAb prevents the hypoglycaemia and increased muscle F2,6BP levels caused in mice by TNF . In African children with severe malaria, hypoglycaemia is associated with very high circulating concentrations of TNF . These observations are consistent with a role for MIF in the pathogenesis of malarial hypoglycaemia through the excessive enhancement of glycolysis in skeletal muscle. The presence of MIF in skeletal muscle would, by inhibiting the suppressive effects of glucocorticoids on inflammatory cytokine production , allow the observed high iNOS induction (Fig. 4C).
MIF protein has previously been reported in ependymal cells, astrocytes, and subpopulations of neurons in the bovine brain , but no comment was made on its presence or absence in cerebral blood vessel walls. These authors also noted its intranuclear location in several cell types, as we have in skeletal muscle, ependymal cells, and non-cerebral endothelial cells in malaria and sepsis. Nuclear staining of skeletal muscle for MIF was commonly associated with high iNOS in the cytoplasm of these cells. The roles of MIF in nuclei have not yet been determined, the only functional association of its expression in this organelle so far reported being in pulmonary adenocarcinoma cells, where is it is associated with a better clinical outcome . As noted, MIF had an asymmetrical association with iNOS in chest vessel walls. This is consistent with MIF appearing before iNOS, as this literature predicts.
MIF protein has been reported in the endothelial cells of human umbilical vein , but to our knowledge neither the neuropil nor blood vessels of human brains have previously, except for our recent conference proceedings report , been examined for its presence. The absence of MIF staining in the walls of the cerebral vasculature during malaria and bacterial sepsis (Figs 3F & 3H), despite evidence for its strong presence in the rest of the vascular tree, is remarkable. It was also absent in all other brains examined, including an additional 17 from other children dying of various causes in Malawi (data not shown). This absence could help explain previous observations of others. For example, the brain could thereby be innately protected from the excesses of systemic inflammation, in the same way as neutralising anti-MIF antibody protects the whole organism in experimental sepsis . Absence of local vascular wall MIF would mean that glucocorticoids would be locally unopposed, leading to much less local transcription of inflammatory responses in the brain than elsewhere in the body, as reported in DNA microarray studies of the caecal ligation and puncture model of sepsis , another example of systemic inflammation. This is consistent with cerebral vasculature iNOS never, in our experience, achieving densities seen in peripheral vessels (Figs 3C vs 4D).
We suggest two possible reasons why low MIF, and thus, through unrestricted glucocorticoid activity, relatively low iNOS, should be biologically advantageous in the cerebral vasculature. First, since normal brain function requires a subtly triggered network of calcium-controlled generation of NO from constitutive NOS, relatively uncontrolled release of NO through iNOS could override this, with loss of function. Second, we propose that dangerous increases in brain perfusion pressure are minimised through MIF being absent from the cerebral vasculature. The brain is protected from capillary rupture because resistance of larger cerebral arteries is remarkably high, keeping the pressure in intracranial arterioles lower than in similar vessels elsewhere . Endotoxin-induced iNOS diminishes the normal constrictor responses that maintain the normally low cerebral perfusion pressure . Were cerebral vascular iNOS to increase as much during a severe systemic infection as elsewhere in the body, the resistance of cerebral arterioles would decrease accordingly, and the hydrostatic pressure of cerebral capillaries would increase, exposing these vessels to pressure they normally do not experience and cannot withstand, and microhemorrhages would ensue. This could account for the co-localisation of vascular wall iNOS and microhemorrhages in Category C brains. An inability to induce MIF in the cerebral vasculature, leading in turn to less induction of iNOS, may thus have a survival advantage in preventing excessive increases in cerebral perfusion pressure, and the risk of intracranial haemorrhage during systemic infections. This may also explain the observation that endotoxin infusion into human volunteers reduces systemic vascular resistance, but leaves cerebral vascular resistance unaltered .
Since MIF opposes the function of glucocorticoids, and is thus able to upregulate iNOS [47, 48], it was of interest to document the change in iNOS as well as MIF in fatal human malaria and sepsis. Except for its routine presence in human airway epithelium , iNOS is rarely detectable by immunological methods in healthy tissues. For example iNOS has been reported to be absent by immunohistochemistry in normal human blood vessel wall  and by reverse transcriptase-driven in situ polymerase chain reaction none could be detected in human brain [51, 52], and very little in cerebellum or skeletal muscle . Most of these authors have used the main commercial antibody source, Transduction Laboratories, that we have employed. Our argument that we are indeed detecting authentic iNOS is strengthened by detecting the same antigen distribution with three unrelated anti-iNOS antibodies described in Methods.
A third (11/32) of the Malawian children diagnosed as cerebral malaria on the basis of established clinical criteria, including peripheral P falciparum parasitaemia, had neither histological changes, nor more than scant sequestered parasites, in the brain sections we stained for iNOS (Category A in the Table 1). The relative absence of parasites from these tissue sections, even though they were prominent on peripheral blood smears on admission, cannot be explained by their clearance from the brain by antimalarial treatment, since very similar average times (mean +/- SEM 21.2 +/- 4.6 hr for Category A, and 27.0 +/- 6.0 hr for Category B) had lapsed from admission, when blood smears were taken and treatment began, to time of death in both these cases and those in Category B, in which parasites were common in brain sections. In the majority of cases in Category A, iNOS was scanty or undetectable in brain vessels (Fig. 3A). In each patient group there was evident diversity in the quantity of iNOS detectable in both brain and muscle (Table 1). As discussed, MIF differences could contribute to this. Since the local release of infected red cell contents during post-schizogony rupture of erythrocytes is accepted to trigger the inflammatory cascade in malaria, the intensity of staining for iNOS would vary throughout the 48 hr schizogony cycle. Since the tissue sections were collected at variable times during the cycle, some variation in iNOS staining intensity between patients with otherwise similar clinical syndromes is therefore inevitable.
About half of the cerebral malaria cases (Category C, 14/32) showed significant microhemorrhages, intravascular accumulations of mononuclear cells, and at times fibrin. Mononuclear cell accumulations (Fig. 2H) have been described previously in cerebral malaria brains from Indian adults  and Thai adults [55, 56], but not, so far as we are aware, African children. These features were negligible in the brain sections examined from the other cerebral malaria categories. In contrast to the low iNOS seen in cerebral vessel walls in Categories A and B, all except three of the 14 brains in Category C scored 3+ or 4+. It is possible that mononuclear cells, fibrin and parasitised red cells could all have contributed to local ischaemia by reducing blood flow through affected vessels, and the consequent ischaemia could, in turn, have contributed to iNOS increase . However, in view of the negligible iNOS induction in Category B brains, despite intense sequestration, it seems more likely that the iNOS induction in vascular walls uniquely observed in Category C brains has resulted from the strong local stimulus provided by recent rupture of schizont-infected red cells. Nevertheless, histological staining cannot establish a chain of cause and effect; this awaits other types of experimentation.
High iNOS concentrations in cerebral vessel walls in Category C was almost invariably associated with equally strong, and often stronger, staining in chest wall vasculature and skeletal muscle (and elsewhere; unpublished data). The virtual absence of sequestered parasites in chest wall sections with intense iNOS staining (Figs 2D and 2E) suggests that a circulating iNOS inducer, such as TNF, may be responsible. The intense iNOS staining in striated muscle from Category B patients who had extensive brain sequestration but no or little iNOS, may similarly indicate a circulating stimulus to muscle iNOS in severe malaria in the absence of a local stimulus in the brain. This stimulus could have systemic metabolic consequences that contribute to altered consciousness through mechanisms other than the local cerebral events observed histologically in Category C cases. Our data thus support the observation that the syndrome diagnosed clinically as cerebral malaria may be the result of a variety of different underlying events , some being local within the brain, and others a metabolic disorder resulting from the systemic infection .
The intense staining for iNOS in cerebral vessel walls that characterise Category C brains could have local pathological consequences. NO generated by the constitutive forms of NOS performs many essential signalling and regulatory roles in the brain, and these normally are precisely controlled by cytoplasmic calcium levels. Examples include the regulation of cerebral blood flow , N-methyl-D-aspartate (NMDA) receptors , and the induction of sleep when release occurs in the pedunculopontine tegmentum . NO also inhibits the Na+/K+ ATPase-driven pump [63, 64] that prevents sodium and therefore water accumulation, or oedema, in various tissues, including brain. These neuronal homeostatic functions of NO can be expected to be altered when iNOS is induced in the brain in high concentrations, as we describe in some of these cases. For example, it has been proposed that the reduced uptake of glutamate by astrocytes when inflammatory cytokine levels are high, allowing this excitotoxin to build up to levels that can cause seizures, is mediated by the NO that these cytokines induce . In addition, vasodilation from the NO so generated could, from animal studies , lower the high cerebral vascular resistance that normally protects the cerebral capillaries from rupture .
Levels of nitrogen oxides, indirect measures of total NO production, in plasma [66, 67], urine or cerebrospinal fluid  have not been associated with degree of coma in severe falciparum malaria. However, total circulating nitrite plus nitrate merely gives an indication of total NO production in the whole body, and is too blunt an instrument to measure the local concentration of a molecule that acts within a cell or two of its site of production. The total NO production in the body's large mass of skeletal muscle, as implied by the degree of iNOS induction in muscles we have observed, could be sufficient to obscure the contribution to plasma levels by the cerebral vasculature. Likewise, our arguments for pathogenic roles for iNOS-induced NO in severe falciparum malaria do not conflict with studies on an iNOS promoter polymorphism associated with increased NO production and protection from severe malaria in East African children , since a population study gives no evidence against these molecules being deleterious in individual fatal cases. In an additional study to that reported here we have found nitrotyrosine where iNOS is detectable, suggesting that NO is actually generated by this enzyme.
A group of 10 comatose children were found, after admission into the study, to be carrying only few or no malaria parasites (and thus were not diagnosed as cerebral malaria), and pathogenic bacteria were cultured from the blood of 5 of them (Table 1). As noted, their cerebral vascular walls contained little iNOS, although iNOS was evident in skeletal muscle and the walls of blood vessels within it. In Category A cerebral malaria cases, the brain vessels were similarly devoid of parasites or iNOS staining. These patients also suffered a fatal comatose illness, suggesting that cerebral vascular wall NO is not necessary for encephalopathy to develop in systemic infections. The high iNOS in the chest wall vessels and skeletal muscle of some of the culture-positive sepsis cases (Figs 3C and 3D), as well as our Category A cerebral malaria cases (Figs 2B,2C,2D,2E), suggests that death may have been due to systemic inflammation rather than intracerebral mechanisms. It is also consistent with the high nitrite/nitrate levels recently reported in skeletal muscle of fatal, but not non-fatal, sepsis cases .
The considerable induction of iNOS in skeletal muscle in a number of our malaria and sepsis patients is consistent with parallels in their pathophysiology . Lanone and co-workers  recently made the first report of iNOS in skeletal muscle in human sepsis. We can confirm their findings (Fig. 3D), and extend it to falciparum malaria (Fig. 2E). From the recognised effects of iNOS-induced NO in skeletal muscle and diaphragm , such NO could cause poor contractility of the ventilatory muscles, and thus contribute to the terminal respiratory arrest observed in severe falciparum malaria by others in African children . Because of a need to hyperventilate to compensate for metabolic acidosis, a common condition in severe malaria in Malawian children , their respiratory muscles would be particularly vulnerable to the effect of this iNOS.