Browsing by Author "Tran, Tuan M."
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Item 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.Item A Psychometric Evaluation of the NIH Toolbox Fluid Cognition Tests Adapted for Swahili and Dholuo Languages in Kenyan Children and Adolescents(Cambridge University Press, 2023) McHenry, Megan S.; Roose, Anna; Abuonji, Emily; Nyalumbe, Mark; Ayuku, David; Ayodo, George; Tran, Tuan M.; Kaat, Aaron J.; Pediatrics, School of MedicineObjective: Our objective was to evaluate the psychometric properties of the culturally adapted NIH Toolbox African Languages® when used in Swahili and Dholuo-speaking children in western Kenya. Method: Swahili-speaking participants were recruited from Eldoret and Dholuo-speaking participants from Ajigo; all were <14 years of age and enrolled in primary school. Participants completed a demographics questionnaire and five fluid cognition tests of the NIH Toolbox® African Languages program, including Flanker, Dimensional Change Card Sort (DCCS), Picture Sequence Memory, Pattern Comparison, and List Sorting tests. Statistical analyses examined aspects of reliability, including internal consistency (in both languages) and test-retest reliability (in Dholuo only). Results: Participants included 479 children (n = 239, Swahili-speaking; n = 240, Dholuo-speaking). Generally, the tests had acceptable psychometric properties for research use within Swahili- and Dholuo-speaking populations (mean age = 10.5; SD = 2.3). Issues related to shape identification and accuracy over speed limited the utility of DCCS for many participants, with approximately 25% of children unable to match based on shape. These cultural differences affected outcomes of reliability testing among the Dholuo-speaking cohort, where accuracy improved across all five tests, including speed. Conclusions: There is preliminary evidence that the NIH Toolbox ® African Languages potentially offers a valid assessment of development and performance using tests of fluid cognition in Swahili and Dholuo among research settings. With piloting underway across other diverse settings, future research should gather additional evidence on the clinical utility and acceptability of these tests, specifically through the establishment of norming data among Kenyan regions and evaluating these psychometric properties.Item Accumulation of Neutrophil Phagocytic Antibody Features Tracks With Naturally Acquired Immunity Against Malaria in Children(Oxford University Press, 2023) Nziza, Nadege; Tran, Tuan M.; DeRiso, Elizabeth A.; Dolatshahi, Sepideh; Herman, Jonathan D.; de Lacerda, Luna; Junqueira, Caroline; Lieberman, Judy; Ongoiba, Aissata; Doumbo, Safiatou; Kayentao, Kassoum; Traore, Boubacar; Crompton, Peter D.; Alter, Galit; Medicine, School of MedicineBackground: Studies have demonstrated the protective role of antibodies against malaria. Young children are known to be particularly vulnerable to malaria, pointing to the evolution of naturally acquired clinical immunity over time. However, whether changes in antibody functionality track with the acquisition of naturally acquired malaria immunity remains incompletely understood. Methods: Using systems serology, we characterized sporozoite- and merozoite-specific antibody profiles of uninfected Malian children before the malaria season who differed in their ability to control parasitemia and fever following Plasmodium falciparum (Pf) infection. We then assessed the contributions of individual traits to overall clinical outcomes, focusing on the immunodominant sporozoite CSP and merozoite AMA1 and MSP1 antigens. Results: Humoral immunity evolved with age, with an expansion of both magnitude and functional quality, particularly within blood-stage phagocytic antibody activity. Moreover, concerning clinical outcomes postinfection, protected children had higher antibody-dependent neutrophil activity along with higher levels of MSP1-specific IgG3 and IgA and CSP-specific IgG3 and IgG4 prior to the malaria season. Conclusions: These data point to the natural evolution of functional humoral immunity to Pf with age and highlight particular antibody Fc-effector profiles associated with the control of malaria in children, providing clues for the design of next-generation vaccines or therapeutics.Item Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria(Rockefeller University Press, 2019-04-12) Hart, Geoffrey T.; Tran, Tuan M.; Theorell, Jakob; Schlums, Heinrich; Arora, Gunjan; Rajagopalan, Sumati; Sangala, A. D. Jules; Welsh, Kerry J.; Traore, Boubacar; Pierce, Susan K.; Crompton, Peter D.; Bryceson, Yenan T.; Long, Eric O.; Medicine, School of MedicineHow antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum–infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum–infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.Item Association of severe malaria with cognitive and behavioural outcomes in low- and middle-income countries: a meta-analysis and systematic review(BMC, 2023-08-03) Ssemata, Andrew Sentoogo; Nakitende, Ann Jacquelline; Kizito, Simon; Thomas, Melissa R.; Islam, Sumaiya; Bangirana, Paul; Nakasujja, Noeline; Yang, Ziyi; Yu, Yunpeng; Tran, Tuan M.; John, Chandy C.; McHenry, Megan S.; Social and Behavioral Sciences, School of Public HealthBackground: Malaria affects 24 million children globally, resulting in nearly 500,000 child deaths annually in low- and middle-income countries (LMICs). Recent studies have provided evidence that severe malaria infection results in sustained impairment in cognition and behaviour among young children; however, a formal meta-analysis has not been published. The objective was to assess the association between severe malaria infection with cognitive and behavioural outcomes among children living in LMICs. Methods: Six online bibliographic databases were searched and reviewed in November 2022. Studies included involved children < 18 years of age living in LMICs with active or past severe malaria infection and measured cognitive and/or behaviour outcomes. The quality of studies was assessed. Definitions of severe malaria included cerebral malaria, severe malarial anaemia, and author-defined severe malaria. Results from all studies were qualitatively summarized. For studies with relevant data on attention, learning, memory, language, internalizing behaviour and externalizing behaviour, results were pooled and a meta-analysis was performed. A random-effects model was used across included cohorts, yielding a standardized mean difference between the severe malaria group and control group. Results: Out of 3,803 initial records meeting the search criteria, 24 studies were included in the review, with data from 14 studies eligible for meta-analysis inclusion. Studies across sub-Saharan Africa assessed 11 cohorts of children from pre-school to school age. Of all the studies, composite measures of cognition were the most affected areas of development. Overall, attention, memory, and behavioural problems were domains most commonly found to have lower scores in children with severe malaria. Meta-analysis revealed that children with severe malaria had worse scores compared to children without malaria in attention (standardized mean difference (SMD) -0.68, 95% CI -1.26 to -0.10), memory (SMD -0.52, 95% CI -0.99 to -0.06), and externalizing behavioural problems (SMD 0.45, 95% CI 0.13-0.78). Conclusion: Severe malaria is associated with worse neuropsychological outcomes for children living in LMICs, specifically in attention, memory, and externalizing behaviours. More research is needed to identify the long-term implications of these findings. Further interventions are needed to prevent cognitive and behavioural problems after severe malaria infection.Item Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages(American Society for Microbiology, 2018-09-21) Clemente, Tatiana M.; Mulye, Minal; Justis, Anna V.; Nallandhighal, Srinivas; Tran, Tuan M.; Gilk, Stacey D.; Microbiology and Immunology, School of MedicineCoxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.Item Decoding the complexities of human malaria through systems immunology(Wiley, 2020-01) Tran, Tuan M.; Crompton, Peter D.; Medicine, School of MedicineThe complexity of the Plasmodium parasite and its life cycle poses a challenge to our understanding of the host immune response against malaria. Studying human immune responses during natural and experimental Plasmodium infections can enhance our understanding of malaria-protective immunity and inform the design of disease-modifying adjunctive therapies and next-generation malaria vaccines. Systems immunology can complement conventional approaches to facilitate our understanding of the complex immune response to the highly dynamic malaria parasite. In this review, recent studies that used systems-based approaches to evaluate human immune responses during natural and experimental Plasmodium falciparum and Plasmodium vivax infections as well as during immunization with candidate malaria vaccines are summarized and related to each other. The potential for next-generation technologies to address the current limitations of systems-based studies of human malaria are discussed.Item Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host(American Society for Microbiology, 2019-06-18) Griesenauer, Brad; Tran, Tuan M.; Fortney, Kate R.; Janowicz, Diane M.; Johnson, Paula; Gao, Hongyu; Barnes, Stephen; Wilson, Landon S.; Liu, Yunlong; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineA major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi Our results suggest that H. ducreyi survives in an abscess by utilizing l-ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection.Item Differential Recruitment of Host Proteins to the Coxiella Burnetii Vacuole in the Absence of the Sterol Reductase CBU1206(2020-08) Ratnayake, Rochelle Chashmi; Gilk, Stacey; Yang, X. Frank; Tran, Tuan M.; Sullivan, William J., Jr.Q fever is a heavily underdiagnosed and underreported infection caused by the obligate intracellular pathogen Coxiella burnetii. Following entry into the host cell, Coxiella replicates in the acidic phagolysosome-like parasitophorous vacuole termed the Coxiella Containing Vacuole (CCV). The CCV is a large and highly fusogenic compartment that actively fuses with the host endocytic pathway during maturation of the phagolysosome. Evidence suggests that the development of the CCV is sensitive to increasing cholesterol levels and leads to CCV acidification and bacterial death. Therefore, we hypothesize that CCV cholesterol concentration is carefully modulated through the Coxiella encoded sterol reductases (CBU1206 and CBU1158). A ∆CBU1206 mutant of Coxiella is hypersensitive to cholesterol and displays growth defects in intracellular replication and CCV development. Following fusion with the host endocytic pathway, the Coxiella NMII Phase II (WT) CCVs readily acquire host proteins such as LAMP1, CD63, Rab7, ORP1L, RILP, and LC3. These heterotypic events with the host endosomal cascade are presumed to provide selected subsets of endocytosed cargo and membrane. Therefore, I investigated whether ΔCBU1206 CCV heterotypic fusion events are defective due to altered lipid content on the CCV membrane. I observed increased accumulation of sterols on the ΔCBU1206 CCV membrane. Similar to WT, the mutant readily fuses host lysosomes and readily acquires the host glycoprotein LAMP1 but displays reduced localization of CD63 (LAMP3). Additionally, reduced localization of the late endosomal markers Rab7, ORP1L, and RILP was observed suggesting that late endosome fusion maybe defective in ΔCBU1206. Further, reduced localization of LC3 was also observed suggesting that the mutant may also be defective in fusing with autophagosomes. Finally, the mutant possesses a functional Type 4 Secretion System that secretes a moderate amount of effector proteins relative to WT. Considering the vast array of functions accomplished by the effectors secreted, the moderate effector secretion by the mutant could influence the endocytic pathway fusion processes as well as CCV development. Collectively, this body of work suggests that the lack of sterol reductase CBU1206 in Coxiella results in defective heterotypic fusion events of the CCV membrane that could alter pathogenesis and CCV expansion.Item Evaluation of an ultrasensitive HRP2-based rapid diagnostic test for detection of asymptomatic Plasmodium falciparum parasitaemia among children in western Kenya(BMC, 2022-11-16) Turnbull, Lindsey B.; Ayodo, George; Knight, Veronicah; John, Chandy C.; McHenry, Megan S.; Tran, Tuan M.; Medicine, School of MedicineBackground Accurate detection of asymptomatic malaria parasitaemia in children living in high transmission areas is important for malaria control and reduction programmes that employ screen-and-treat surveillance strategies. Relative to microscopy and conventional rapid diagnostic tests (RDTs), ultrasensitive RDTs (us-RDTs) have demonstrated reduced limits of detection with increased sensitivity to detect parasitaemia in symptomatic individuals. In this study, the performance of the NxTek™ Eliminate Malaria P.f test was compared with traditional microscopy and quantitative polymerase chain reaction (qPCR) testing methods of detection for P. falciparum parasitaemia among asymptomatic children aged 7–14 years living in an area of high malaria transmission intensity in western Kenya. Methods In October 2020, 240 healthy children without any reported malaria symptoms were screened for the presence of P. falciparum parasitaemia; 120 children were randomly selected to participate in a follow-up visit at 6–10 weeks. Malaria parasitaemia was assessed by blood-smear microscopy, us-RDT, and qPCR of a conserved var gene sequence from genomic DNA extracted from dried blood spots. Sensitivity, specificity, and predictive values were calculated for field diagnostic methods using qPCR as the gold standard. Comparison of detectable parasite density distributions and area under the curve were also calculated to determine the effectiveness of the us-RDT in detecting asymptomatic infections with low parasite densities. Results The us-RDT detected significantly more asymptomatic P. falciparum infections than microscopy (42.5% vs. 32.2%, P = 0.002). The positive predictive value was higher for microscopy (92.2%) than for us-RDT (82.4%). However, false negative rates were high for microscopy and us-RDT, with negative predictive values of 53.7% and 54.6%, respectively. While us-RDT detected significantly more infections than microscopy overall, the density distribution of detectable infections did not differ (P = 0.21), and qPCR detected significantly more low-density infections than both field methods (P < 0.001, for both comparisons). Conclusions Us-RDT is more sensitive than microscopy for detecting asymptomatic malaria parasitaemia in children. Though the detectable parasite density distributions by us-RDT in our specific study did not significantly differ from microscopy, the additional sensitivity of the us-RDT resulted in more identified asymptomatic infections in this important group of the population and makes the use of the us-RDT advisable compared to other currently available malaria field detection methods.