allopurinol and Malaria--Falciparum

The emergence of chloroquine resistance, and a world-wide scarcity of quinine, have resulted in a search for newer antimalarial drugs directed against falciparum malaria. Allopurinol causes virtually complete inhibition of purine biosynthesis of malaria parasites, which may prove lethal to the parasites. This study was designed to examine if allopurinol is additive to quinine in the treatment of acute falciparum malaria. Forty-seven Asian-Indian adults with acute complicated falciparum malaria were assigned to a treatment period of five days. They were randomly assigned to receive either oral allopurinol (12 mg/kg in three divided doses for five days) plus quinine (600 mg intravenously every 8 hr for two days, followed by 600 mg orally every 8 hr for three days ) (n = 24), or quinine alone (600 mg intravenously every 8 hr for two days, followed by 600 mg orally every 8 hr for three days) (n = 23). The responses were assessed by parasite clearance time, defervescence time, splenomegaly disappearance time, and cure rate. In the allopurinol-quinine (ALLQUIN)-treated group, all the durations were significantly shorter than those in the quinine alone (QUIN)-treated group. They were ALLQUIN versus QUIN (mean +/- SD = 65.33 +/- 11.47 hr versus 76.78 +/- 18.20 hr; P = 0.0214; 57.66 +/- 13.01 hr versus 82.52 +/- 23.55 hr, P = 0.0002; 10 +/- 1.64 days versus 14.65 +/- 2.4 days; P = 0.0002), respectively. The cure rate was higher in the ALLQUIN group (91.7%) than in the QUIN group (87%). However, this difference was not statistically significant. Therefore, this study indicates that allopurinol can be an additive to quinine to bring about both faster eradication of Plasmodium falciparum and clinical remission than with quinine alone.

Topics: Acute Disease; Acute Kidney Injury; Adolescent; Adult; Aged; Allopurinol; Anemia; Antimalarials; Antimetabolites; Drug Therapy, Combination; Female; Follow-Up Studies; Humans; Malaria, Cerebral; Malaria, Falciparum; Male; Middle Aged; Parasitemia; Quinine; Splenomegaly

Severe anemia is an important contributor to mortality in children with severe malaria. Anemia in malaria is a multi-factorial complication, since dyserythropoiesis, hemolysis and phagocytic clearance of uninfected red blood cells (RBCs) can contribute to this syndrome. High levels of oxidative stress and immune dysregulation have been proposed to contribute to severe malarial anemia, facilitating the clearance of uninfected RBCs. In a cohort of 552 Ugandan children with severe malaria, we measured the levels of xanthine oxidase (XO), an oxidative enzyme that is elevated in the plasma of malaria patients. The levels of XO in children with severe anemia were significantly higher compared to children with severe malaria not suffering from severe anemia. Levels of XO were inversely associated with RBC hemoglobin (ρ =  - 0.25, p < 0.0001), indicating a relation between this enzyme and severe anemia. When compared with the levels of immune complexes and of autoimmune antibodies to phosphatidylserine, factors previously associated with severe anemia in malaria patients, we observed that XO is not associated with them, suggesting that XO is associated with severe anemia through an independent mechanism. XO was associated with prostration, acidosis, jaundice, respiratory distress, and kidney injury, which may reflect a broader relation of this enzyme with severe malaria pathology. Since inhibitors of XO are inexpensive and well-tolerated drugs already approved for use in humans, the validation of XO as a contributor to severe malarial anemia and other malaria complications may open new possibilities for much needed adjunctive therapy in malaria.

Topics: Anemia; Antigen-Antibody Complex; Child; Erythrocytes; Humans; Malaria, Falciparum; Xanthine Oxidase

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1β, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.

Topics: Caspase 1; Cells, Cultured; Cytokines; Host-Pathogen Interactions; Humans; Inflammation; Inflammation Mediators; Macrophage Activation; Macrophages; Malaria, Cerebral; Malaria, Falciparum; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Plasmodium falciparum; Reactive Oxygen Species; Signal Transduction; Xanthine Oxidase

Infection by Plasmodium falciparum is the leading cause of malaria in humans. The parasite contains a unique and essential plastid-like organelle called the apicoplast that, similar to the mitochondria and chloroplast, houses its own genome that must undergo replication and repair. The putative apicoplast replicative DNA polymerase, POM1, has no direct orthologs in mammals, making the P. falciparum POM1 an attractive antimalarial drug target. Here, we report on a fluorescent high-throughput DNA polymerase assay that relies on the ability of POM1 to perform strand-displacement synthesis through the stem of a DNA hairpin substrate, thereby separating a Cy3 dye from a quencher. Assay-validation experiments were performed using 384-well plates and resulted in a signal window of 7.90 and aZ' factor of 0.71. A pilot screen of a 2880-compound library identified 62 possible inhibitors that cause more than 50% inhibition of polymerase activity. The simplicity and statistical robustness of the assay suggest it is well suited for the screening of novel apicoplast polymerase inhibitors that may serve as lead compounds in antimalarial drug-discovery efforts.

Topics: Antimalarials; Apicoplasts; Chloroplasts; DNA; DNA-Directed DNA Polymerase; Drug Discovery; Exonucleases; Humans; Kinetics; Malaria, Falciparum; Mitochondria; Multienzyme Complexes; Nucleic Acid Synthesis Inhibitors; Peptide Library; Plasmodium falciparum; Protozoan Proteins; Spectrometry, Fluorescence

The role of proinflammatory cytokine production in the pathogenesis of malaria is well established, but the identification of the parasite products that initiate inflammation is not complete. Hemozoin is a crystalline metabolite of hemoglobin digestion that is released during malaria infection. In the present study, we characterized the immunostimulatory activity of pure synthetic hemozoin (sHz) in vitro and in vivo. Stimulation of naive murine macrophages with sHz results in the MyD88-independent activation of NF-kappaB and ERK, as well as the release of the chemokine MCP-1; these responses are augmented by IFN-gamma. In macrophages prestimulated with IFN-gamma, sHz also results in a MyD88-dependent release of TNF-alpha. Endothelial cells, which encounter hemozoin after schizont rupture, respond to sHz by releasing IL-6 and the chemokines MCP-1 and IL-8. In vivo, the introduction of sHz into the peritoneal cavity produces an inflammatory response characterized by neutrophil recruitment and the production of MCP-1, KC, IL-6, IL-1alpha, and IL-1beta. MCP-1 and KC are produced independently of MyD88, TLR2/4 and TLR9, and components of the inflammasome; however, neutrophil recruitment, the localized production of IL-1beta, and the increase in circulating IL-6 require MyD88 signaling, the IL-1R pathway, and the inflammasome components ICE (IL-1beta-converting enzyme), ASC (apoptosis-associated, speck-like protein containing CARD), and NALP3. Of note, inflammasome activation by sHz is reduced by allopurinol, which is an inhibitor of uric acid synthesis. These data suggest that uric acid is released during malaria infection and may serve to augment the initial host response to hemozoin via activation of the NALP3 inflammasome.

Topics: Allopurinol; Animals; Apoptosis Regulatory Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins; Chemokine CCL2; Chemokine CXCL1; Cytoskeletal Proteins; Endothelial Cells; Extracellular Signal-Regulated MAP Kinases; Hemeproteins; Inflammation; Interferon-gamma; Interleukin-1alpha; Interleukin-1beta; Interleukin-6; Interleukin-8; Macrophages; Malaria, Falciparum; Mice; Myeloid Differentiation Factor 88; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Plasmodium falciparum; Signal Transduction; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Uric Acid

Elevated plasma levels of xanthine oxidase and liver function parameters have been associated with inflammatory events in several human diseases. While xanthine oxidase provides in vitro protection against malaria, its pathophysiological functions in vivo and interactions with liver function parameters remain unclear. This study examined the interactions and plasma levels of xanthine oxidase (XO) and uric acid (UA), catalase (CAT) and liver function parameters GOT, GPT and bilirubin in asymptomatic (n=20), uncomplicated (n=32), and severe (n=18) falciparum malaria children aged 3-13 years. Compared to age-matched control (n=16), significant (p<0.05) elevation in xanthine oxidase by 100-550%, uric acid by 15.4-153.8%, GOT and GPT by 22.1-102.2%, and total bilirubin by 2.3-86% according to parasitaemia (geometric mean parasite density (GMPD)=850-87100 parasites/microL) was observed in the malarial children. Further comparison with control revealed higher CAT level (16.2+/-0.5 vs 14.6+/-0.4 U/L; p<0.05) lacking significant (p>0.05) correlation with XO, but lower CAT level (13.4-5.4 U/L) with improved correlations (r=-0.53 to -0.91; p<0.05) with XO among the asymptomatic and symptomatic malaria children studied. 75% of control, 45% of asymptomatic, 21.9% of uncomplicated, and none of severe malaria children had Hb level>11.0 g/dL. Multivariate analyses further revealed significant (p<0.05) correlations between liver function parameters and xanthine oxidase (r=0.57-0.64) only in the severe malaria group. We conclude that elevated levels of XO and liver enzymes are biochemical features of Plasmodium falciparum parasitaemia in Nigerian children, with both parameters interacting differently to modulate the catalase response in asymptomatic and symptomatic falciparum malaria.

Topics: Adolescent; Alanine Transaminase; Animals; Aspartate Aminotransferase, Mitochondrial; Bilirubin; Catalase; Child; Child, Preschool; Female; Hemoglobins; Humans; Liver; Liver Function Tests; Malaria, Falciparum; Male; Multivariate Analysis; Nigeria; Parasitemia; Plasmodium falciparum; Uric Acid; Xanthine Oxidase