Browsing by Author "Gopinadhan, Adnan"

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    A Human Pluripotent Stem Cell-Derived In Vitro Model of the Blood-Brain Barrier in Cerebral Malaria
    (2024-01) Gopinadhan, Adnan; John, Chandy C.; Nelson, David E.; Bauer, Margaret E.; Absalon, Sabrina; Tran, Tuan M.
    Blood-brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but the physiological relevance remains uncertain. I aimed to develop a novel in vitro model of the BBB in CM using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) that mimic a near in vivo barrier phenotype. hiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 hours. Barrier integrity was measured using transendothelial electrical resistance (TEER). Localization and expression of tight junction (TJ) proteins, occludin and zona occludin-1 (ZO-1), and endothelial marker, intercellular adhesion molecule 1 (ICAM-1) was determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were also measured. hiPSC-BMECs showed improved barrier integrity and localization of TJ proteins compared to immortalized BMECs. After 6-hours of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and disruption of TJ protein localization compared to co-culture with uninfected RBCs (RBCs), but no change in TJ protein expression was observed by WB in the Pf-iRBCs co-cultures. Expression of ICAM-1 on hiPSC-BMECs co-cultured with Pf-iRBCs was higher compared to co-culture with RBCs. In addition, there was an increase in expression of the angiogenin, platelet factor 4, and phospho-heat shock protein-27 in the Pf-iRBCs co-cultures compared to co-cultures with RBCs. These findings demonstrate the physiological relevance of our hiPSC-BMEC-based in vitro model of the BBB, as determined by elevated TEER and appropriate TJ protein localization. In co-culture with Pf-iRBCs, breakdown in the barrier integrity, changes in TJ protein localization, increase in expression of ICAM-1, and of markers of angiogenesis and cellular stress, all point towards a more relevant in vitro model, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.
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    A human pluripotent stem cell-derived in vitro model of the blood-brain barrier in cerebral malaria
    (Springer Nature, 2024-05-01) Gopinadhan, Adnan; Hughes, Jason M.; Conroy, Andrea L.; John, Chandy C.; Canfield, Scott G.; Datta, Dibyadyuti; Pediatrics, School of Medicine
    Background: Blood-brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties. Methods: hiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays. Results: After 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture. Conclusion: These findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.
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    Blood biomarkers of neuronal injury in paediatric cerebral malaria and severe malarial anaemia
    (Oxford University Press, 2023-11-27) Datta, Dibyadyuti; Gopinadhan, Adnan; Soto, Alejandro; Bangirana, Paul; Opoka, Robert O.; Conroy, Andrea L.; Saykin, Andrew J.; Kawata, Keisuke; John, Chandy C.; Pediatrics, School of Medicine
    Persistent neurodisability is a known complication in paediatric survivors of cerebral malaria and severe malarial anaemia. Tau, ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein have proven utility as biomarkers that predict adverse neurologic outcomes in adult and paediatric disorders. In paediatric severe malaria, elevated tau is associated with mortality and neurocognitive complications. We aimed to investigate whether a multi-analyte panel including ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein can serve as biomarkers of brain injury associated with mortality and neurodisability in cerebral malaria and severe malarial anaemia. In a prospective cohort study of Ugandan children, 18 months to 12 years of age with cerebral malaria (n = 182), severe malarial anaemia (n = 158), and asymptomatic community children (n = 118), we measured admission blood levels of ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein. We investigated differences in biomarker levels, associations with mortality, blood–brain barrier integrity, neurodeficits and cognitive Z-scores in survivors up to 24-month follow-up. Admission ubiquitin C-terminal hydrolase-L1 levels were elevated >95th percentile of community children in 71 and 51%, and neurofilament-light chain levels were elevated >95th percentile of community children in 40 and 37% of children with cerebral malaria and severe malarial anaemia, respectively. Glial fibrillary acidic protein was not elevated in disease groups compared with controls. In cerebral malaria, elevated neurofilament-light chain was observed in 16 children who died in hospital compared with 166 survivors (P = 0.01); elevations in ubiquitin C-terminal hydrolase-L1 levels were associated with degree of blood–brain barrier disruption (P = 0.01); and the % predictive value for neurodeficits over follow-up (discharge, 6-, 12-, and 24 months) increased for ubiquitin C-terminal hydrolase-L1 (60, 67, 72, and 83), but not neurofilament-light chain (65, 68, 60, and 67). In cerebral malaria, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse memory scores in children <5 years at malaria episode who crossed to over 5 years old during follow-up cognitive testing [β −1.13 (95% confidence interval −2.05, −0.21), P = 0.02], and elevated neurofilament-light chain was associated with worse attention in children ≥5 years at malaria episode and cognitive testing [β −1.08 (95% confidence interval −2.05, −1.05), P = 0.03]. In severe malarial anaemia, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse attention in children <5 years at malaria episode and cognitive testing [β −0.42 (95% confidence interval −0.76, −0.07), P = 0.02]. Ubiquitin C-terminal hydrolase-L1 and neurofilament-light chain levels are elevated in paediatric cerebral malaria and severe malarial anaemia. In cerebral malaria, elevated neurofilament-light chain is associated with mortality whereas elevated ubiquitin C-terminal hydrolase-L1 is associated with blood–brain barrier dysfunction and neurodeficits over follow-up. In cerebral malaria, both markers are associated with worse cognition, while in severe malarial anaemia, only ubiquitin C-terminal hydrolase-L1 is associated with worse cognition.
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    Cerebrospinal fluid biomarkers provide evidence for kidney-brain axis involvement in cerebral malaria pathogenesis
    (Frontiers Media, 2023-05-02) Conroy, Andrea L.; Datta, Dibyadyuti; Opoka, Robert O.; Batte, Anthony; Bangirana, Paul; Gopinadhan, Adnan; Mellencamp, Kagan A.; Akcan-Arikan, Ayse; Idro, Richard; John, Chandy C.; Pediatrics, School of Medicine
    Introduction: Cerebral malaria is one of the most severe manifestations of malaria and is a leading cause of acquired neurodisability in African children. Recent studies suggest acute kidney injury (AKI) is a risk factor for brain injury in cerebral malaria. The present study evaluates potential mechanisms of brain injury in cerebral malaria by evaluating changes in cerebrospinal fluid measures of brain injury with respect to severe malaria complications. Specifically, we attempt to delineate mechanisms of injury focusing on blood-brain-barrier integrity and acute metabolic changes that may underlie kidney-brain crosstalk in severe malaria. Methods: We evaluated 30 cerebrospinal fluid (CSF) markers of inflammation, oxidative stress, and brain injury in 168 Ugandan children aged 18 months to 12 years hospitalized with cerebral malaria. Eligible children were infected with Plasmodium falciparum and had unexplained coma. Acute kidney injury (AKI) on admission was defined using the Kidney Disease: Improving Global Outcomes criteria. We further evaluated blood-brain-barrier integrity and malaria retinopathy, and electrolyte and metabolic complications in serum. Results: The mean age of children was 3.8 years (SD, 1.9) and 40.5% were female. The prevalence of AKI was 46.3% and multi-organ dysfunction was common with 76.2% of children having at least one organ system affected in addition to coma. AKI and elevated blood urea nitrogen, but not other measures of disease severity (severe coma, seizures, jaundice, acidosis), were associated with increases in CSF markers of impaired blood-brain-barrier function, neuronal injury (neuron-specific enolase, tau), excitatory neurotransmission (kynurenine), as well as altered nitric oxide bioavailability and oxidative stress (p < 0.05 after adjustment for multiple testing). Further evaluation of potential mechanisms suggested that AKI may mediate or be associated with CSF changes through blood-brain-barrier disruption (p = 0.0014), ischemic injury seen by indirect ophthalmoscopy (p < 0.05), altered osmolality (p = 0.0006) and through alterations in the amino acids transported into the brain. Conclusion: In children with cerebral malaria, there is evidence of kidney-brain injury with multiple potential pathways identified. These changes were specific to the kidney and not observed in the context of other clinical complications.
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    Investigating the presence of Pfkelch gene mutations in Ugandan children with severe malaria
    (2017) Gopinadhan, Adnan; Chandy, John; Alexander, Dent; Tuan, Tran
    Artemisinin resistance was first observed in Southeast Asia (SEA) and could pose a threat to malaria treatment all over the world. Recently mutations in the propeller region of Pfkelch13 gene have been used as a genetic marker for resistance observed in SEA. We investigated the presence of mutations in the Pfkelch gene in children in Kampala, Uganda with severe malaria (SM) treated with intravenous quinine, or with asymptomatic P.falciparum infection (AP) treated with artemether-lumefantrine. We sequenced the Pfkelch gene (2178bp) in 157 children with SM and 49 children with AP infection. In children with SM and AP we identified 106 (60.8%) and 27 (55.1%) parasites with mutations upstream of the Pfkelch13 propeller region. The two most prevalent mutations were 142NN (26.1% in SM, 33% in AP) and K189T (16.5% in SM, 12.2% in AP). In SM, only a single infection had a mutation in the propeller region (A578S), while in AP, mutations in the propeller region included A578S (n=1) and S522C (n=1). In children with SM, parasites with 142NN insertion compared to 3D7 Pfkelch13 parasites had lower parasite density (p=0.02) and lower parasite biomass (p=0.03). Children with SM who either had 142NN or K189T mutation cleared parasites after quinine treatment faster than those with the 3D7 Pfkelch13 genotype (P<0.001 for both mutations compared to 3D7). In this cohort mutations, upstream of the Pfkelch13 propeller region were common. Future studies will assess the presence of Pfcrt and Pfmdr mutations in this cohort, and how these relate to the Pfkelch13 mutations and to parasite clearance.
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    Multiplexed and High-Throughput Label-Free Detection of RNA/Spike Protein/IgG/IgM Biomarkers of SARS-CoV-2 Infection Utilizing Nanoplasmonic Biosensors
    (American Chemical Society, 2021-06-29) Masterson, Adrianna N.; Muhoberac, Barry B.; Gopinadhan, Adnan; Wilde, David J.; Deiss, Frédérique T.; John, Chandy C.; Sardar, Rajesh; Chemistry and Chemical Biology, School of Science
    To tackle the COVID-19 outbreak, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an unmet need for highly accurate diagnostic tests at all stages of infection with rapid results and high specificity. Here, we present a label-free nanoplasmonic biosensor-based, multiplex screening test for COVID-19 that can quantitatively detect 10 different biomarkers (6 viral nucleic acid genes, 2 spike protein subunits, and 2 antibodies) with a limit of detection in the aM range, all within one biosensor platform. Our newly developed nanoplasmonic biosensors demonstrate high specificity, which is of the upmost importance to avoid false responses. As a proof of concept, we show that our detection approach has the potential to quantify both IgG and IgM antibodies directly from COVID-19-positive patient plasma samples in a single instrument run, demonstrating the high-throughput capability of our detection approach. Most importantly, our assay provides receiving operating characteristics, areas under the curve of 0.997 and 0.999 for IgG and IgM, respectively. The calculated p-value determined through the Mann-Whitney nonparametric test is <0.0001 for both antibodies when the test of COVID-19-positive patients (n = 80) is compared with that of healthy individuals (n = 72). Additionally, the screening test provides a calculated sensitivity (true positive rate) of 100% (80/80), a specificity (true negative rate) >96% (77/80), a positive predictive value of 98% at 5% prevalence, and a negative predictive value of 100% at 5% prevalence. We believe that our very sensitive, multiplex, high-throughput testing approach has potential applications in COVID-19 diagnostics, particularly in determining virus progression and infection severity for clinicians for an appropriate treatment, and will also prove to be a very effective diagnostic test when applied to diseases beyond the COVID-19 pandemic.