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Abstract Background Malaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host’s immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines. Methods The sample group comprised 33 patients admitted with malaria caused by Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation. Results At admission, patients with P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level (rs = −0.630, p = 0.038). However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria. Conclusions This is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria.

Abstract Background The efficacy of anti-malarial drugs is determined by the level of parasite susceptibility, anti-malarial drug bioavailability and pharmacokinetics, and host factors including immunity. Host immunity improves the in vivo therapeutic efficacy of anti-malarial drugs, but the mechanism and magnitude of this effect has not been characterized. This study characterized the effects of 'immune' plasma to Plasmodium falciparumon the in vitro susceptibility of P. falciparum to anti-malarial drugs. Methods Titres of antibodies against blood stage antigens (mainly the ring-infected erythrocyte surface antigen [RESA]) were measured in plasma samples obtained from Thai patients with acute falciparum malaria. 'Immune' plasma was selected and its effects on in vitro parasite growth and multiplication of the Thai P. falciparum laboratory strain TM267 were assessed by light microscopy. The in vitro susceptibility to quinine and artesunate was then determined in the presence and absence of 'immune' plasma using the 3H-hypoxanthine uptake inhibition method. Drug susceptibility was expressed as the concentrations causing 50% and 90% inhibition (IC50 and IC90), of 3H-hypoxanthine uptake. Results Incubation with 'immune' plasma reduced parasite maturation and decreased parasite multiplication in a dose dependent manner. 3H-hypoxanthine incorporation after incubation with 'immune' plasma was decreased significantly compared to controls (median [range]; 181.5 [0 to 3,269] cpm versus 1,222.5 [388 to 5,932] cpm) (p= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC50 6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (p = 0.02), and also had a borderline effect on artesunate susceptibility; IC50 0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (p = 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC90 values were not significantly affected; median (range) IC90 448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (p > 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (p = 0.4). Conclusions 'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent in vitro anti-malarial drug susceptibility of P. falciparum. The IC90 was much less affected than the IC50 measurement.

Abstract Background Severe and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in Plasmodium falciparum malaria. Methods The utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral (CM; n = 87) and severe (non-cerebral) malaria (SM; n = 36) from uncomplicated malaria (UM; n = 70). Comparative statistics are reported using a non-parametric univariate analysis (Kruskal-Wallis test or Chi-squared test, as appropriate). Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups (UM, SM, CM), adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma. Results ANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates (ethnicity, age, parasitaemia, sex). Further, there was a significant decrease in ANG-1 levels in patients with SM (non-cerebral) versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM (area under the curve, p-value: ANG-2, 0.763, p < 0.001; ANG-1, 0.884, p < 0.001; Ratio, 0.857, p < 0.001) or UM and CM (area under the curve, p-value: ANG-2, 0.772, p < 0.001; ANG-1, 0.778, p < 0.001; Ratio, 0.820, p < 0.001). Conclusions These results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes.

Abstract Background It has frequently been reported that Plasmodium vivax suppressed Plasmodium falciparum and ameliorated disease severity in patients infected with these two species simultaneously. The authors investigate the hypothesis that immunological responses stimulated by P. vivax may play a role in suppressing co-infecting P. falciparum. Methods Sera, taken sequentially from one of the authors (YN) during experimental infection with P. vivax, were added to in vitro cultures of P. falciparum. Cross-reactive antibodies against P. falciparum antigens, and cytokines were measured in the sera. Results Significant growth inhibitory effects upon P. falciparum cultures (maximally 68% inhibition as compared to pre-illness average) were observed in the sera collected during an acute episode. Such inhibitory effects showed a strong positive temporal correlation with cross-reactive antibodies, especially IgM against P. falciparum schizont extract and, to a lesser degree, IgM against Merozoite Surface Protein (MSP)-119. Interleukin (IL)-12 showed the highest temporal correlation with P. vivax parasitaemia and with body temperatures in the volunteer. Conclusion These results suggest the involvement by cross-reactive antibodies, especially IgM, in the interplay between plasmodial species. IL-12 may be one of direct mediators of fever induction by rupturing P. vivax schizonts, at least in some subjects. Future studies, preferably of epidemiological design, to reveal the association between cross-reactive IgM and cross-plasmodial interaction, are warranted.

Abstract Introduction Oxidative stress has been demonstrated in malaria. The potential oxidative modification of lipoproteins derived from malaria patients was studied. These oxidized lipids may have role in pathogenesis of malaria. Method The plasma lipid profile and existence of oxidized forms of very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were investigated in malaria (17 mild and 24 severe patients) and 37 control subjects. Thiobarbituric acid reactive substances (TBARs), conjugated dienes, tryptophan fluorescence and fluidity of lipoproteins were determined as markers of oxidation. The biological effect of malarial lipoproteins was assessed by the expression of adhesion molecules on endothelial cells. Results Malarial lipoproteins had decreased cholesterol (except in VLDL) and phospholipid. The triglyceride levels were unchanged. The cholesterol/phospholipid ratio of LDL was decreased in malaria, but increased in VLDL and HDL. TBARs and conjugate dienes were increased in malarial lipoproteins, while the tryptophan fluorescence was decreased. The fluidity of lipoproteins was increased in malaria. These indicated the presence of oxidized lipoproteins in malaria by which the degree of oxidation was correlated with severity. Of three lipoproteins from malarial patients, LDL displayed the most pronounced oxidative modification. In addition, oxidized LDL from malaria patients increased endothelial expression of adhesion molecules. Conclusion In malaria, the lipoproteins are oxidatively modified, and the degree of oxidation is related with severity. Oxidized LDL from malarial patients increases the endothelial expression of adhesion molecules. These suggest the role of oxidized lipoproteins, especially LDL, on the pathogenesis of disease.