Immunology Dept, NMIMR

Background: Cerebral malaria (CM) is responsible for most of the malaria-related deaths in children in sub-Saharan Africa. Although, not well understood, the pathogenesis of CM involves parasite and host factors which contribute to parasite sequestration through cytoadherence to the vascular endothelium. Cytoadherence to brain microvasculature is believed to involve host endothelial receptor, CD54 or intercellular adhesion molecule (ICAM)-1, while other receptors such as CD36 are generally involved in cytoadherence of parasites in other organs. We therefore investigated the contributions of host ICAM-1 expression and levels of antibodies against ICAM-1 binding variant surface antigen (VSA) on parasites to the development of CM.

The surface marker patterns of T cells of Ghanaian children during measles infection were studied and an attempt was made to demonstrate T cell activation and viability in vitro after activation in vivo by measles virus. The frequencies of CD4 + and CD8 + naive T cells in measles patients were high while their memory T cells were remarkably reduced with no sign of proliferation even at the acute phase of the illness. The reduction of memory T cells was prolonged during the convalescent phase (2 months after onset). The anti-CD3 monoclonal antibody-induced expression of interleukin-2 receptor a chain (IL-2WCD25) was significantly suppressed; however, the addition of phorbol 12-myristate 13-acetate or ionomycin caused a remarkable recovery of CD25 expression. On simple culture, an appreciable proportion of T cells from measles patients died rapidly in contrast with only a few T cells from healthy controls doing so. The suppression of CD25 expression was still demonstrated during the convalescent phase of the disease. Taken together these results suggest unresponsiveness and activation-induced cell death of T cells during severe measles infection in Ghanaian children. Furthermore the prolonged abnormalities of T cells (i.e. decreased memory T cells and inhibition of CD25 expression during the convalescent phase) might be related to postmeasles infection immunosuppressive status.

To assess the eosinophil response to Plasmodium falciparum infection a cohort of initially parasite-free Ghanaian children was followed for 3 months. Seven of nine children who acquired an asymptomatic P. falciparum infection showed increase in eosinophil counts, while a decrease was found in seven of nine children with symptomatic malaria, and no change was observed in 14 children who remained parasitefree. In a hospital-based study, paediatric patients with cerebral malaria (CM), severe anaemia (SA), or uncomplicated malaria (UM) had uniformly low eosinophil counts during the acute illness followed by eosinophilia 30 days after cure. Plasma levels of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured as indicators of eosinophil activation. In spite of the low eosinophil counts, ECP levels were increased on day 0 and significantly higher in patients with CM (geometric mean (95% confidence interval) 8·5 ng/ml (6·8-10·7 ng/ml)) than in SA (4·7 ng/ml (3·0-7·5 ng/ml)) and UM patients (4·3 ng/ml (3·6-5·3 ng/ml), P < 0·001). A similar pattern was found for EPX. It thus appears that the low eosinophil counts may be due to tissue sequestration and destruction rather than decreased production. The plasma levels of the granule proteins correlated with levels of tumour necrosis factor and soluble IL-2 receptor, implicating inflammatory responses and T cell activation as causes of the eosinophil activation. By contrast, the eosinophil induction did not appear to be part of a Th2-like response. Eosinophil granule proteins may be important in both control of malaria infection and the pathogenesis of severe malaria.

Slaughterhouse surveys are important in the detection and management of zoonotic diseases. Routine reports from the Kumasi slaughterhouse, in the Ashanti region of Ghana, include cases of zoonotic diseases. Due to its location and size, Kumasi is the major cattle market and an important transit point for cattle trade from places within and outside Ghana. This present study was designed to examine slaughterhouse reports and to explore the nature of the knowledge, attitude and practices of butchers who operate at this slaughterhouse, in relation to zoonoses. The study was largely descriptive, employing qualitative methods and tools. Butchers were interviewed and their practices along the production line observed. The study indicates that zoonotic diseases are frequently detected at the Kumasi slaughterhouse. However the knowledge, attitudes, practices and beliefs of the butchers are largely inadequate for their profession in view of the important public health role that butchers play. The butchers have never received any form of training. It is recommended that the butchers receive training on a regular basis and that laws be formulated and implemented to protect the health of the butchers and the general public.

Levels of soluble CD30 (sCD30) in serum were elevated in patients with Plasmodium falciparum malaria but showed decline following treatment. The levels of sCD30 in serum were correlated significantly with the expression of gamma interferon by peripheral T cells. These data suggest that CD30 ؉ cells are upregulated during a malaria attack and that they may play a regulating role at the site of inflammation.

A prospective study that included 429 children for active detection of mild malaria was conducted in a coastal region of Ghana to reveal whether the incidence of malaria is affected by human leukocyte antigen (HLA) polymorphism. During 12 months of follow-up, 85 episodes of mild clinical malaria in 74 individuals were observed, and 34 episodes among them were accompanied with significant parasitemia at &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;5000 infected red blood cells per cubic millimeter. Attributable and relative risks conferred by genetic factors in the HLA region were evaluated by comparison of the incidence in children, stratified by carrier status, of a given allele of HLA-A, -B, -DRB1 and TNFA promoter polymorphism. HLA-B*35:01 reduced the incidence by 0.178 events per person per year (0.060 versus 0.239 for B*35:01-positive and -negative subpopulations, respectively), and a relative risk of 0.25, which remained statistically significant after Bonferroni&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s correction for multiple testing (p(c) = 8.2 × 10(-5)). Further, HLA-B*35:01 and -B*53:01 exhibited opposite effects on the incidence of malaria with significant parasitemia. When parasite densities in different HLA carriers status were compared, HLA-A*01 conferred an increase in parasite load (p = 6.0 × 10(-7)). In addition, we found a novel DRB1 allele that appears to have emerged from DRB1*03:02 by single nucleotide substitution.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of infected erythrocytes. Each parasite genome contains about 40 PfEMP1 genes, but only 1 PfEMP1 gene is expressed at a given time. PfEMP1 serves as a parasite-sequestering ligand to endothelial cells and enables the parasites to avoid splenic passage. PfEMP1 antibodies may protect from disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen. In this study, we have measured antibodies in Ghanaian children to a conserved region of PfEMP1 by enzyme-linked immunosorbent assay and antibodies to variant molecules on erythrocytes infected with field isolates of P. falciparum by flow cytometry. Based on close clinical monitoring, the children were grouped into those who did (susceptible) and those who did not (protected) have malaria during the season. The prevalences of antibodies to both the conserved PfEMP1 peptide and the variant epitopes were greater than 50%, and the levels of immunoglobulin G (IgG) correlated with age. The levels of antibodies to both the conserved peptide and the variant epitopes were higher in protected than in susceptible children. After correcting for the effect of age, the levels of IgG to variant antigens on a Sudanese and a Ghanaian parasite isolate remained significantly higher in protected than in susceptible children. Thus, the levels of IgG to variant antigens expressed on the surface of infected erythrocytes correlated with protection from clinical malaria. In contrast, the levels of IgG to a peptide derived from a conserved part of PfEMP1 did not correlate with protection from malaria.

This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the block 2 region of MSP-1 were measured in a cohort of 280 children before the beginning of the major malaria transmission season. The cohort was then actively monitored for malaria, clinically and parasitologically, over a period of 17 months. Evidence is presented for an association between antibody responses to block 2 and a significantly reduced risk of subsequent clinical malaria. Furthermore, statistical survival analysis provides new information on the duration of the effect over time. The results support a conclusion that the block 2 region of MSP-1 is a target of protective immunity against P. falciparum and, thus, a promising new candidate for the development of a malaria vaccine.

␥␦ T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the ␥␦ 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 ␥␦ 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 ␥␦ T cells involved in this perturbation expressed V␦1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the V␦1 ؉ cell population at the peak of their increase showed that all expressed V␥ chains were used, and CDR3 length polymorphism indicated that the expanded V␦1 population was highly polyclonal. A very high proportion of the V␦1 ؉ T cells produced gamma interferon, while fewer V␦1 ؉ cells than the average proportion of all CD3 ؉ cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-␥␦ ؉ cells in general or V␦1 ؉ cells in particular. Taken together, our data point to an immunoregulatory role of the expanded V␦1 ؉ T-cell population in this group of semi-immune P. falciparum malaria patients.

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. 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 (&amp;amp;amp;gt;110 g/L), moderate anaemia (60-90 g/L), and severe anaemia (&amp;amp;amp;lt;50 g/L). IL-10 and TNF concentrations were measured by ELISA in plasma samples from all patients. 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&amp;amp;amp;lt;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&amp;amp;amp;lt;0.001). In both studies, TNF concentrations were significantly higher in cerebral malaria than in fully conscious patients (p&amp;amp;amp;lt;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&amp;amp;amp;lt;0.001). 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.