Faculty Bibliography

BACKGROUND:Cerebral malaria is a complication of Plasmodium falciparum infections where the integrity of the blood-brain barrier (BBB) is compromised, frequently leading to persistent neurological sequelae or death. The current treatment for cerebral malaria is based on rapid elimination of the parasite with intravenous anti-malarial drugs, but there is no adjunctive treatment that could address the loss of BBB integrity, potentially decreasing sequelae and improving the survival rate. Prenylation, a cellular process that incorporates isoprenoid lipids to specific protein sites, is known to regulate endothelial barrier integrity. Here we have studied the role of the isoprenoid farnesyl pyrophosphate (farnesyl-PP) and of different inhibitors of the enzymes that perform prenylation in endothelial cells to determine their effect on the endothelial barrier disruption induced by Plasmodium falciparum. METHODS:An in vitro model using human brain microvascular endothelial cells (HBMECs) incubated with lysates of P. falciparum-infected red blood cells (iRBCs) and a mouse model of cerebral malaria were used to study the role of the two main prenylation enzymes, farnesyltransferase and geranylgeranyltransferase, in the regulation of endothelial barrier integrity in relation to cerebral malaria. RESULTS:Addition of exogenous farnesyl-PP to HBMECs incubated with P. falciparum-iRBCs resulted in increased endothelial barrier disruption. Conversely, treatment with inhibitors of farnesyltransferase, but not of geranylgeranyltransferase, resulted in protection of barrier integrity against disruption induced by P. falciparum-iRBCs. Mice infected with Plasmodium berghei that were treated with the farnesyltransferase inhibitor tipifarnib presented delayed onset of cerebral malaria and higher survival rates. CONCLUSIONS:Inhibitors of farnesyltransferase protect human endothelial cell barrier integrity from disruption induced by P. falciparum in vitro and decrease mortality in mice with experimental cerebral malaria, indicating a role for farnesylation in the regulation of barrier integrity during cerebral malaria and identifying a potential drug target for the protection of the endothelium during severe malaria.

In this work, the synthesis and structural characterization of coordination complexes C1 (Co-Cl), C2 (Cu-Cl), C3 (Co-Br), and C4 (Ni-Cl) with the ligand 2-(tert-butoxy)-6-(1H-imidazol-1-yl)pyridine (L) was carried out. The X-ray diffraction reveals the presence of four equatorial L and two axial halogens as coligands. The synthesized complexes crystallize in a P-1 space group. Topological analysis using ToposPro assigned the discrete 1,6M7-1 topology, in which ZA1 and ZA2 represent nonequivalent supramolecular ligand contacts, while the suffix -1 denotes the first nonisomorphic net not included in the RCSR database. The complexes were evaluated against Plasmodium falciparum, showing the high influence of the metal coordination in the modulation of the antiplasmodial activity relative to the free ligand, particularly against the resistant phenotype. All evaluated complexes retained inhibitory effects against both chloroquine-sensitive and chloroquine-resistant P. falciparum strains. Among the series, C3 and C4 emerged as the most promising candidates due to their favorable balance between antiplasmodial activity, selectivity, and low hemolytic profile. Computational analyses further revealed that modulation of the electronic properties, chemical softness, and lipophilic profiles of the complexes may contribute to their distinct biological behavior, while molecular docking suggested plausible interactions with parasite-associated enzymatic targets.

This work describes the synthesis, characterization, and preliminary evaluation of the anti-Plasmodium falciparum activity of a copper(II) coordination-complex derived from an imidazole-based ligand. This compound was characterized by Infrared, UV-Vis, Raman, high resolution mass spectrometry, fluorescence, Thermogravimetric analysis, and electrical conductivity allowing the elucidation of their structure and which was also confirmed by single-crystal X-ray diffraction. From the crystallographic analysis, it was found that the copper atom is situated in an octahedral environment, with both carboxylate ligands occupying equatorial positions and the ligands L located axially. Interestingly, the acetate ions act as bidentate ligands, but in an anisobidentate binding mode, where the oxygen atoms exhibit different bond distances to the metal center, resulting in asymmetric coordination. This phenomenon is attributed to strong hydrogen bonding within the crystal packing. Biological assays performed to test the complex revealed low hemocytotoxicity, with values ≤20% at concentrations above 500 µg/mL. When evaluated in vitro against Plasmodium falciparum (3D7 strain, chloroquine-sensitive), the complex exhibited significantly higher activity than the free ligand. These findings indicate that coordination to the metal center plays a substantial role in enhancing antiparasitic-activity, providing a solid foundation for future investigations aimed at elucidating the underlying mechanisms and advancing research on the anti-Plasmodium falciparum activity of metal complexes.

Cerebral malaria is a major complication of Plasmodium falciparum infection that occurs upon the sequestration of infected red blood cells (iRBCs) in brain capillaries, resulting in the loss of endothelial barrier integrity, brain swelling, and frequently long-term sequelae or death. P. falciparum-iRBCs cause the disruption of human brain microvascular endothelial cell barrier integrity in vitro, mimicking the microenvironment of cerebral malaria, yet the specific mechanisms mediating this process remain unknown. Upon infection of the host RBCs, P. falciparum produces hemozoin, a crystal form of heme generated following the degradation of hemoglobin by the parasite. Here, we show that the endothelial barrier-disrupting activity is found entirely in the hemozoin fraction of P. falciparum-iRBCs. This activity is not caused by the hemozoin crystal itself, which is not able to induce barrier disruption, but by the biomolecules that are associated with it. Treatment of purified P. falciparum hemozoin with proteases inhibits the disruption of endothelial barrier integrity caused by the hemozoin, indicating an important role for proteins in the disruption of the barrier. Conversely, treatment with nucleases did not affect hemozoin barrier-disrupting activity. These results identify a key molecular mechanism in the P. falciparum-mediated brain endothelial barrier disruption during cerebral malaria and may open new avenues for the treatment of this complication.IMPORTANCEWhile several specific biomolecules have been proposed to contribute to the disruption of endothelial barrier integrity in cerebral malaria, no single Plasmodium falciparum- or host-derived factor has been definitively identified as the primary driver of this disruption. Here, we identify the brain endothelial barrier-disruptive P. falciparum-infected red blood cell (iRBC)-derived activity to be caused by biomolecules bound to hemozoin, identifying a key, novel mechanism in the pathogenesis of cerebral malaria. The finding that P. falciparum hemozoin also disrupts a pulmonary endothelial cell barrier opens the possibility that this mechanism underlies other severe malaria complications. The implication of P. falciparum-iRBC-derived proteins in this mechanism is in line with previous reports, providing a novel interpretation of these findings in the context of hemozoin-binding. This knowledge provides a new perspective in the search for specific molecules and mechanisms involved in barrier disruption, which may lead to the development of much-needed specific treatments for cerebral malaria.

BACKGROUND:Cerebral malaria (CM) is a serious complication of Plasmodium falciparum malaria that causes coma and frequently death in patients. In malaria-endemic settings, a large percentage of the population presents with incidental P. falciparum malaria parasitemia. In the absence of specific biomarkers for CM, when these individuals suffer from bacterial or viral infections causing coma, they are frequently misdiagnosed with CM. METHODS:We have tested the specificity for CM of two candidate biomarkers for severe malaria, Angiopoietin-like 4 and Inhibin-βE, which are secreted by endothelial cells in response to P. falciparum infected erythrocytes. The levels of these biomarkers were determined retrospectively in the plasma of a cohort of 379 Kenyan children including cases of severe malaria caused by CM, respiratory distress or severe anemia, as well as cases of non-traumatic coma of unknown cause. RESULTS:Angiopoietin-like 4 and Inhibin-βE showed high specificity for severe malaria, including CM (AUC 0.82), respiratory distress (AUC 0.86) and severe anemia (AUC 0.85), when compared to acute non-traumatic coma of non-malarial etiology. Specificity was further increased when the biomarkers were used in combination with platelet levels (AUC 0.96). Angiopoietin-like 4 and Inhibin-βE are also predictors of death by CM (AUC 0.85). CONCLUSIONS:Angiopoietin-like 4 and Inhibin-βE could be developed as a diagnostic tool for the differentiation of comatose patients with CM from other causes of coma.

INTRODUCTION/UNASSIGNED:The SARS-CoV-2 virus resulted in significant disability and diagnostic challenges among patients with Post-Acute Sequelae of COVID-19 (PASC). Here, we assessed microvascular perfusion, clotting, and autoimmune responses to lung targets in PASC patients compared to healthy controls with the aim of explaining the persistent respiratory symptoms of patients with PASC. METHODS/UNASSIGNED:We performed a blinded case-control study of 20 PASC patients with persistent respiratory symptoms versus 20 healthy controls previously infected with SARS-CoV-2 virus. We assessed lung perfusion using Technetium-99m macroaggregated albumin (MAA) SPECT-CT scans, clotting using coagulation and thromboelastrogram (TEG) tests, and autoimmunity to vascular and lung antigens using ELISA assays. RESULTS/UNASSIGNED:Subjective respiratory symptoms and quality-of-life measures were significantly worse among the PASC patients compared with healthy controls (p<0.001). Clinical symptoms among PASC patients were inversely correlated with plasma total IgA levels (coefficient: -0.61, p=0.004) and with autoimmune IgA recognizing pulmonary microvascular endothelial cell antigens (coefficient: -0.51, p=0.02). Additionally, levels of total IgA were directly correlated with fibrinogen and fibrin-related clot strength (coefficient: +0.52, p=0.02; coefficient: +0.63, p=0.003). SPECT-CT scans were positive only among 25% of PASC cases versus 10% of healthy controls (p=0.41). TEG tests showed no differences between the groups. CONCLUSIONS/UNASSIGNED:Our small study of PASC patients identified that circulating IgA antibodies may correlate inversely with clinical symptoms and directly with clotting parameters, suggesting a possible link between autoimmunity and coagulation. However, many of the study's findings were null, which may mean that tissue-level studies or alternative explanations of PASC need to be explored.

Infection with Plasmodium falciparum carries a significant risk of cerebral malaria (CM). Children are particularly susceptible to human CM (HCM) which manifests as an acute neurovascular encephalopathy leading to high levels of mortality. Occurring in parallel with CM, malarial retinopathy (MR) is readily detected on ophthalmoscopy as one or more of: white-centered retinal hemorrhage, retinal whitening, and vessel discoloration. It leads to several distinct types of blood retinal barrier (BRB) breakdown. The precise molecular mechanisms underpinning CM and MR remain ill-defined, but parasitemia is known to drive progressive neurovascular obstruction and inflammation leading to cerebral and retinal edema and ischemia. Extensive clinical studies in patients with CM have shown that retinal examination is a useful approach for understanding pathology and an indicator for risk of mortality and morbidity. Fully understanding the cellular and molecular mechanisms that underpin CM and MR is important for developing new therapeutic approaches and in this regard the murine model of experimental CM (ECM) has proved to offer considerable value. Much is known about brain pathology in this model although much less is understood about the retina. In this review, we seek to evaluate MR in clinical scenarios and make comparisons with the retina from mice with ECM. Through detailed in vivo and post-mortem studies in the mouse and human retina, this review highlights the links between CM and MR and how this will aid our understanding of the disease progression and pathogenesis.

We investigated the role of uric acid in the pathogenesis of severe malaria (SM) in two independent cohorts of children with SM. Hyperuricemia (blood uric acid ≥ 7 mg dl^-1) was present in 25% of children with SM and was associated with increased in-hospital mortality and postdischarge mortality in both cohorts. Increased blood uric acid levels were also associated with worse scores in overall cognition in children with SM < 5 years old in both cohorts. Hemolysis of infected red blood cells and impaired renal excretion of uric acid were the primary drivers of hyperuricemia in SM. Hyperuricemia was associated with multiple complications of SM, including acute kidney injury, acidosis, impaired perfusion, coma and intestinal injury with increases in the abundance of Gram-negative uricase-producing pathobionts (Escherichia and Shigella) in the stool. Clinical trials evaluating uric acid-lowering medications as adjunctive therapy for children with SM should be considered to improve survival and protect neurodevelopment.

Neglected tropical diseases such as Chagas disease, human African trypanosomiasis, leishmaniasis, and schistosomiasis have a significant global health impact in predominantly developing countries, although these diseases are spreading due to increased international travel and population migration. Drug repurposing with a focus on increasing antiparasitic potency and drug-like properties is a cost-effective and efficient route to the development of new therapies. Here we identify compounds that have potent activity against Trypanosoma cruzi and Leishmania donovani, and the latter were progressed into the murine model of infection. Despite the potent in vitro activity, there was no effect on parasitemia, necessitating further work to improve the pharmacokinetic properties of this series. Nonetheless, valuable insights have been obtained into the structure-activity and structure-property relationships of this compound series.

BACKGROUND:Recently, there has been an unexplained increase in the incidence of blackwater fever (BWF) in Eastern Uganda. In this study, we evaluate the association between immune complexes, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and the occurrence and recurrence of BWF in children with severe malaria (SM). METHODS:Between 2014 and 2017, children aged six months to <4 years hospitalized with SM and community children (CC) were recruited at two hospitals in Central and Eastern Uganda. We measured serum circulating immune complexes (cIC) and their relationship to SM complications and post-discharge outcomes and evaluated effect mediation through G6PD deficiency. RESULTS:557 children with SM and 101 CC were enrolled. The mean age of children was 2.1 years. Children with SM had higher cIC levels than CC, p<0.001. After controlling for age, sex, and site, cIC were associated with severe anemia, jaundice, and BWF (adjusted odds ratio, 95% confidence interval: 7.33 (3.45, 15.58), p<0.0001; 4.31 (1.68, 11.08), p=0.002; and 5.21 (2.06, 13.18), p<0.0001), respectively. cIC predicted readmissions for SM, severe anemia, and BWF (adjusted incidence rate ratios (95% confidence interval): 2.11 (1.33, 3.34), p=0.001; 8.62 (2.80, 26.59), p<0.0001; and 7.66 (2.62, 22.45), p<0.0001), respectively. The relationship was most evident in males where the frequency of the G6PD African allele (A-) was 16.8%. G6PD deficiency was associated with increases in cIC in males (p=0.01) and mediation analysis suggested G6PD deficiency contributes to recurrent severe anemia and BWF via increased cIC. CONCLUSIONS:Immune complexes are associated with hemolytic complications and predict recurrences in SM survivors.