Desperately Seeking Therapies for Cerebral Malaria.

Malaria is a deadly infectious disease caused by parasites of the Plasmodium spp. that takes an estimated 435,000 lives each year, primarily among young African children. For most children, malaria is a febrile illness that resolves with time, but in ∼1% of cases, for reasons we do not understand, malaria becomes severe and life threatening. Cerebral malaria (CM) is the most common form of severe malaria, accounting for the vast majority of childhood deaths from malaria despite highly effective antiparasite chemotherapy. Thus, CM is one of the most prevalent lethal brain diseases, and one for which we have no effective therapy. CM is, in part, an immune-mediated disease, and to fully understand CM, it is essential to appreciate the complex relationship between the malarial parasite and the human immune system. In this study, we provide a primer on malaria for immunologists and, in this context, review progress identifying targets for therapeutic intervention.

MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

A cryptophane-based "turn-on" 129Xe NMR biosensor for monitoring calmodulin.

We present the first cryptophane-based "turn-on" 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.

A "Smart" ¹²8Xe NMR Biosensor for pH-Dependent Cell Labeling.

Here we present a "smart" xenon-129 NMR biosensor that undergoes a peptide conformational change and labels cells in acidic environments. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide that is α-helical at pH 5.5 and disordered at pH 7.5. The (129)Xe NMR chemical shift at room temperature was strongly pH-dependent (Δδ = 3.4 ppm): δ = 64.2 ppm at pH 7.5 vs δ = 67.6 ppm at pH 5.5, where Trp(peptide)-cryptophane interactions were evidenced by Trp fluorescence quenching. Using hyper-CEST NMR, we probed peptidocryptophane detection limits at low-picomolar (10(-11) M) concentration, which compares favorably to other NMR pH reporters at 10(-2)-10(-3) M. Finally, in biosensor-HeLa cell solutions, peptide-cell membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-(129)Xe NMR chemical shift relative to pH 7.5 studies. This highlights new uses for (129)Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for pH-dependent cell labeling in cancer diagnosis and treatment.

Cryptophane-folate biosensor for (129)xe NMR.

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized (129)Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized (129)Xe NMR experiments.

DON in pediatric cerebral malaria, a phase I/IIA dose-escalation safety study: study protocol for a clinical trial.

BACKGROUND: Despite treatment with highly effective antimalarial drugs, malaria annually claims the lives of over half a million children under 5-years of age in sub-Saharan Africa. Cerebral malaria (CM), defined as Plasmodium falciparum infection with coma, is the severe malaria syndrome with the highest mortality. Studies in the CM mouse model suggest that a T cell-mediated response underlies CM pathology, opening a new target for therapy in humans. This trial aims to establish the preliminary safety of one such novel therapy, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). METHODS: In this phase I/IIa dose-escalation clinical trial, a single dose of intravenous (IV) DON is administered to three participants groups-healthy adults and adults with uncomplicated malaria, then pediatric participants with CM-to primarily assess safety. The secondary objective of this trial is to assess pharmacokinetics of DON over a range of doses. The open-label adult portion of the trial enrolls 40 healthy adults concurrently with 40 adults with uncomplicated malaria. Cohorts of 10 participants receive a single IV dose of DON with doses escalating between cohorts from 0.1 mg/kg, 1.0 mg/kg, 5.0 mg/kg, to 10 mg/kg. Following subsequent safety review, a randomized, double-blind, and placebo-controlled pediatric study enrolls 72 participants aged 6 months to 14 years with CM. The pediatric portion of the study minimally spans three malaria seasons including a planned interim analysis after 50% of pediatric enrollments. The first half of pediatric participants receive DON 0.1 mg/kg, 1.0 mg/kg, or placebo. Dosing for the second half of pediatric participants is informed by the safety and preliminary efficacy results of those previously enrolled. The pediatric portion of the study has an exploratory outcome evaluating the preliminary efficacy of DON. Efficacy is assessed by diagnostics predictive of CM outcome: electroencephalography (EEG), magnetic resonance imaging (MRI), and transcranial doppler (TCD), measured before and after DON administration. All participants with malaria receive standard of care antimalarials in accordance with local guidelines, regardless of study drug dose group. DISCUSSION: This preliminary safety and efficacy study evaluates DON, a candidate adjunctive therapy for pediatric CM. If results support DON preliminary safety and efficacy, follow-up phase II and III clinical trials will be indicated. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov on 28 July 2022 (NCT05478720).

CD8+ T cells target cerebrovasculature in children with cerebral malaria.

BACKGROUNDCerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODSUsing multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM-), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV-).RESULTSWe identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM-, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSIONWithin the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDINGThis research was supported by the Intramural Research Program of the National Institutes of Health.

Do we know enough to find an adjunctive therapy for cerebral malaria in African children?

Cerebral malaria is the deadliest complication of malaria, a febrile infectious disease caused by Plasmodium parasite. Any of the five human Plasmodium species can cause disease, but, for unknown reasons, in approximately 2 million cases each year P. falciparum progresses to severe disease, ultimately resulting in half a million deaths. The majority of these deaths are in children under the age of five. Currently, there is no way to predict which child will progress to severe disease and there are no adjunctive therapies to halt the symptoms after onset. Herein, we discuss what is known about the disease mechanism of one form of severe malaria, cerebral malaria, and how we might exploit this understanding to rescue children in the throes of cerebral disease.

Synthesis of enantiopure, trisubstituted cryptophane-A derivatives.

The efficient synthesis of enantiopure, trisubstituted cryptophane-A derivatives, organic host molecules with unusually high xenon affinity, is reported. Synthesis and chromatographic separation of (±) tri-Mosher's acid substituted cryptophane diastereomers gave ready access to the enantiopure cryptophanes, which are critical components in the design of enantiomerically pure (129)Xe biosensors. Hyperpolarized (129)Xe NMR spectroscopy identified single resonances for both trisubstituted cryptophane diastereomers that were separated by 9.5 ppm. This highlights opportunities for using enantiopure xenon biosensors in the simultaneous detection of (129)Xe in different biochemical environments.