Sarah J. Higgins

Combinations of host biomarkers predict mortality among Ugandan children with severe malaria: A retrospective case-control study

Background Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria. Methodology/Findings Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers (angiopoietin-2, soluble ICAM-1, soluble Flt-1, procalcitonin, IP-10, soluble TREM-1) discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity (cross-validated misclassification rate: 15.4%, standard error 4.9%). Conclusions We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria.

Immunopathogenesis of falciparum malaria: implications for adjunctive therapy in the management of severe and cerebral malaria

Despite optimal antimalarial treatment and advances in malaria eradication, the mortality rate associated with severe malaria due to Plasmodium falciparum infection, including cerebral malaria (CM), remains unacceptably high. This suggests that strategies directed solely at parasite eradication may be insufficient to prevent neurological complications and death in all cases of CM. Therefore, there is an urgent need to develop innovative adjunctive therapeutic strategies to effectively reduce CM-associated mortality. CM pathogenesis is believed to be due, in part, to an aberrant host immune response to P. falciparum, resulting in deleterious consequences, including vascular activation and dysfunction. Development of effective and affordable therapeutic strategies that act to modulate the underlying host-mediated immunopathology should be explored to improve outcome. In this article, we summarize immunomodulatory therapies that have been assessed in clinical trials to date, and highlight novel and promising treatment strategies currently being investigated to address this major global health challenge.

Endothelial activation and dysregulation in malaria: a potential target for novel therapeutics.

Purpose of reviewDespite parenteral artesunate therapy, the fatality rate of cerebral malaria remains high. Adjunctive therapy targeting the underlying pathophysiology of cerebral malaria may further improve the clinical outcome. Endothelial activation and dysfunction is a central process in the pathogenesis of cerebral malaria. An improved understanding of how endothelium is perturbed in cerebral malaria may yield novel strategies to diagnose and intervene. Here, we discuss recent findings on the key molecular mediators of endothelial activation/dysregulation in cerebral malaria, and innovative endothelial-based experimental approaches to improve detection and treatment. Recent findingsBiomarkers of endothelial activation [e.g., angiopoietin (Ang)-1, Ang-2, and a soluble form of the Ang-receptor (soluble Tie-2)] have been shown to be reliable predictors of malarial disease severity and mortality, and may improve clinical triage and management. Moreover, they may represent novel therapeutic targets to improve clinical outcome. Restoring bioavailable nitric oxide by administration of inhaled nitric oxide or its substrate, L-arginine, may rescue endothelial function, decrease Ang-2, and improve disease outcome in cerebral malaria. SummaryInterventions targeting the Ang–Tie-2 axis to promote endothelial quiescence, including agents to improve endothelial nitric oxide, represent potential adjunctive therapies for cerebral malaria.

Complement driven innate immune response to malaria: fuelling severe malarial diseases

Severe malaria remains a major cause of global mortality. The innate immune response to infection is a key determinant of malaria severity and outcome. The complement system plays a key role in initiating and augmenting innate immune responses, including inflammation, endothelial activation, opsonization and coagulation, processes which have been implicated in malaria pathogenesis. In this review, we discuss the evidence supporting a role for excessive complement activation in the pathogenesis of severe malaria.

Gene control of tyrosine kinase TIE2 and vascular manifestations of infections

Significance Major infections, such as influenza and bacterial sepsis, kill millions of individuals yearly, most commonly from complications affecting the vasculature, such as acute respiratory distress syndrome. Poor outcomes from rare infections, such as Ebola virus disease, have also been linked to the vasculature. The basis for prominent vascular involvement in infectious syndromes remains poorly understood. The present work shows that humans exhibit common, yet highly consequential, genetic variation in the ability to sustain expression of a key homeostatic vascular receptor called tunica interna endothelial cell kinase 2. The results suggest that host determinants of the molecular vascular response to infection may have a heretofore underappreciated impact on clinical outcomes. They also suggest new means to identify at-risk individuals and personalize future therapies. Ligands of the endothelial-enriched tunica interna endothelial cell kinase 2 (Tie2) are markedly imbalanced in severe infections associated with vascular leakage, yet regulation of the receptor itself has been understudied in this context. Here, we show that TIE2 gene expression may constitute a novel vascular barrier control mechanism in diverse infections. Tie2 expression declined rapidly in wide-ranging models of leak-associated infections, including anthrax, influenza, malaria, and sepsis. Forced Tie2 suppression sufficed to attenuate barrier function and sensitize endothelium to permeability mediators. Rapid reduction of pulmonary Tie2 in otherwise healthy animals attenuated downstream kinase signaling to the barrier effector vascular endothelial (VE)-cadherin and induced vascular leakage. Compared with wild-type littermates, mice possessing one allele of Tie2 suffered more severe vascular leakage and higher mortality in two different sepsis models. Common genetic variants that influence TIE2 expression were then sought in the HapMap3 cohort. Remarkably, each of the three strongest predicted cis-acting SNPs in HapMap3 was also associated with the risk of acute respiratory distress syndrome (ARDS) in an intensive care unit cohort of 1,614 subjects. The haplotype associated with the highest TIE2 expression conferred a 28% reduction in the risk of ARDS independent of other major clinical variables, including disease severity. In contrast, the most common haplotype was associated with both the lowest TIE2 expression and 31% higher ARDS risk. Together, the results implicate common genetic variation at the TIE2 locus as a determinant of vascular leak-related clinical outcomes from common infections, suggesting new tools to identify individuals at unusual risk for deleterious complications of infection.

Dysregulation of angiopoietin-1 plays a mechanistic role in the pathogenesis of cerebral malaria

Angiopoietin-1 is dysregulated in pediatric severe malaria, and targeting it prevents vascular dysfunction and death in a mouse model of cerebral malaria. Bolstering the host to beat malaria Cerebral malaria is a devastating disease associated with high death rates and brain injury despite the use of potent antimalarials. Understanding the role of the host response in determining the ability of the host to survive a severe malaria infection may enable development of host-based therapeutics to prevent infection-induced death. Higgins et al. now demonstrate how the loss of a key host vascular protective protein, angiopoietin-1, is associated with severe and fatal malaria in children and in a mouse model. Administering angiopoietin-1 to mice with cerebral malaria reinforced the blood-brain barrier and improved survival, even when initiated late in disease. Cerebral malaria is a leading cause of global morbidity and mortality. Interventions targeting the underlying pathophysiology of cerebral malaria may improve outcomes compared to treatment with antimalarials alone. Microvascular leak plays an important role in the pathogenesis of cerebral malaria. The angiopoietin (Ang)–Tie-2 system is a critical regulator of vascular function. We show that Ang-1 expression and soluble Tie-2 expression were associated with disease severity and outcome in a prospective study of Ugandan children with severe malaria and in a preclinical murine model of experimental cerebral malaria. Ang-1 was necessary for maintenance of vascular integrity and survival in a mouse model of cerebral malaria. Therapeutic administration of Ang-1 preserved blood-brain barrier integrity and, in combination with artesunate treatment, improved survival beyond that with artesunate alone. These data define a role for dysregulation of the Ang–Tie-2 axis in the pathogenesis of cerebral malaria and support the evaluation of Ang–Tie-2–based interventions as potential adjunctive therapies for treating severe malaria.

KLF2 and KLF4 control endothelial identity and vascular integrity

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal.

Systemic release of high mobility group box 1 (HMGB1) protein is associated with severe and fatal Plasmodium falciparum malaria

BackgroundSevere falciparum malaria (SM) pathogenesis has been attributed, in part, to deleterious systemic host inflammatory responses to infection. High mobility group box 1 (HMGB1) protein is an important mediator of inflammation implicated in sepsis pathophysiology.MethodsPlasma levels of HMGB1 were quantified in a cohort of febrile Ugandan children with Plasmodium falciparum infection, enrolled in a prospective observational case-controlled study, using a commercial enzyme-linked immunosorbent assay. The utility of HMGB1 to distinguish severe malaria (SM; n = 70) from uncomplicated malaria (UM; n = 33) patients and fatal (n = 21) versus non-fatal (n = 82) malaria, at presentation, was examined. Receiver operating characteristic curve analysis was used to assess the prognostic accuracy of HMGB1. The ability of P. falciparum-parasitized erythrocytes to induce HMGB1 from peripheral blood mononuclear cells was assessed in vitro. The effect of an anti-HMGB1 neutralizing antibody on disease outcome was assessed in the experimental Plasmodium berghei ANKA rodent parasite model of SM. Mortality and parasitaemia was assessed daily and compared to isotype antibody-treated controls.ResultsElevated plasma HMGB1 levels at presentation were significantly associated with SM and a subsequent fatal outcome in paediatric patients with P. falciparum infection. In vitro, parasitized erythrocytes induced HMGB1 release from human peripheral blood mononuclear cells. Antibody-mediated neutralization of HMGB1 in the experimental murine model of severe malaria failed to reduce mortality.ConclusionThese data suggest that elevated HMGB1 is an informative prognostic marker of disease severity in human SM, but do not support HMGB1 as a viable target for therapeutic intervention in experimental murine SM.

Tie2 protects the vasculature against thrombus formation in systemic inflammation

Disordered coagulation contributes to death in sepsis and lacks effective treatments. Existing markers of disseminated intravascular coagulation (DIC) reflect its sequelae rather than its causes, delaying diagnosis and treatment. Here we show that disruption of the endothelial Tie2 axis is a sentinel event in septic DIC. Proteomics in septic DIC patients revealed a network involving inflammation and coagulation with the Tie2 antagonist, angiopoietin-2 (Angpt-2), occupying a central node. Angpt-2 was strongly associated with traditional DIC markers including platelet counts, yet more accurately predicted mortality in 2 large independent cohorts (combined N = 1,077). In endotoxemic mice, reduced Tie2 signaling preceded signs of overt DIC. During this early phase, intravital imaging of microvascular injury revealed excessive fibrin accumulation, a pattern remarkably mimicked by Tie2 deficiency even without inflammation. Conversely, Tie2 activation normalized prothrombotic responses by inhibiting endothelial tissue factor and phosphatidylserine exposure. Critically, Tie2 activation had no adverse effects on bleeding. These results mechanistically implicate Tie2 signaling as a central regulator of microvascular thrombus formation in septic DIC and indicate that circulating markers of the Tie2 axis could facilitate earlier diagnosis. Finally, interventions targeting Tie2 may normalize coagulation in inflammatory states while averting the bleeding risks of current DIC therapies.

Host Biomarkers Are Associated With Response to Therapy and Long-Term Mortality in Pediatric Severe Malaria

Background. Host responses to infection are critical determinants of disease severity and clinical outcome. The development of tools to risk stratify children with malaria is needed to identify children most likely to benefit from targeted interventions. Methods. This study investigated the kinetics of candidate biomarkers of mortality associated with endothelial activation and dysfunction (angiopoietin-2 [Ang-2], soluble FMS-like tyrosine kinase-1 [sFlt-1], and soluble intercellular adhesion molecule-1 [sICAM-1]) and inflammation (10 kDa interferon γ-induced protein [CXCL10/IP-10] and soluble triggering receptor expressed on myeloid cells-1 [sTREM-1]) in the context of a randomized, double-blind, placebo-controlled, parallel-arm trial evaluating inhaled nitric oxide versus placebo as adjunctive therapy to parenteral artesunate for severe malaria. One hundred eighty children aged 1–10 years were enrolled at Jinja Regional Referral Hospital in Uganda and followed for up to 6 months. Results. There were no differences between the 2 study arms in the rate of biomarker recovery. Median levels of Ang-2, CXCL10, and sFlt-1 were higher at admission in children who died in-hospital (n = 15 of 180; P < .001, P = .027, and P = .004, respectively). Elevated levels of Ang-2, sTREM-1, CXCL10, and sICAM-1 were associated with prolonged clinical recovery times in survivors. The Ang-2 levels were also associated with postdischarge mortality (P < .0001). No biomarkers were associated with neurodisability. Conclusions. Persistent endothelial activation and dysfunction predict survival in children admitted with severe malaria.