Neutrophils - an understudied bystander in dengue?

Dengue is a mosquito-borne viral disease which causes significant morbidity and mortality each year. Previous research has proposed several mechanisms of pathogenicity that mainly involve the dengue virus and host humoral immunity. However, innate immune cells, such as neutrophils, may also play an important role in dengue, albeit a much less defined role. In this review, we discuss the emerging roles of neutrophils in dengue and their involvement in pathologies associated with severe dengue. We also describe the potential use of several neutrophil proteins as biomarkers for severe dengue. These studies suggest that neutrophils are important players in dengue, and a better understanding of neutrophil-dengue biology is urgently needed.

Study protocol: infectious diseases consortium (I3D) for study on integrated and innovative approaches for management of respiratory infections: respiratory infections research and outcome study (RESPIRO).

BACKGROUND: Community-acquired respiratory infections are a leading cause of illness and death globally. The aetiologies of community-acquired pneumonia remain poorly defined. The RESPIRO study is an ongoing prospective observational cohort study aimed at developing pragmatic logistical and analytic platforms to accurately identify the causes of moderate-to-severe community-acquired pneumonia in adults and understand the factors influencing disease caused by individual pathogens. The study is currently underway in Singapore and has plans for expansion into the broader region. METHODS: RESPIRO is being conducted at three major tertiary hospitals in Singapore. Adults hospitalised with acute community-acquired pneumonia or lower respiratory tract infections, based on established clinical, laboratory and radiological criteria, will be recruited. Over the course of the illness, clinical data and biological samples will be collected longitudinally and stored in a biorepository for future analysis. DISCUSSION: The RESPIRO study is designed to be hypothesis generating, complementary to and easily integrated with other research projects and clinical trials. The detailed clinical database and biorepository will yield insights into the epidemiology and outcomes of community-acquired lower respiratory tract infections in Singapore and the surrounding region and offers the opportunity to deeply characterise the microbiology and immunopathology of community-acquired pneumonia.

Febrile Phase Soluble Urokinase Plasminogen Activator Receptor and Olfactomedin 4 as Prognostic Biomarkers for Severe Dengue in Adults.

BACKGROUND: Dengue cases continue to rise and can overwhelm healthcare systems during outbreaks. In dengue, neutrophil mediators, soluble urokinase plasminogen activator receptor (suPAR) and olfactomedin 4, and mast cell mediators, chymase and tryptase, have not been measured longitudinally across the dengue phases. The utility of these proteins as prognostic biomarkers for severe dengue has also not been assessed in an older adult population. METHODS: We prospectively enrolled 99 adults with dengue-40 dengue fever, 46 dengue with warning signs and 13 severe dengue, along with 30 controls. Plasma levels of suPAR, olfactomedin 4, chymase and tryptase were measured at the febrile, critical and recovery phases in dengue patients. RESULTS: The suPAR levels were significantly elevated in severe dengue compared to the other dengue severities and controls in the febrile (P < .001), critical (P < .001), and recovery (P = .005) phases. In the febrile phase, suPAR was a prognostic biomarker of severe dengue, with an AUROC of 0.82. Using a cutoff derived from Youden's index (5.4 ng/mL) and an estimated prevalence of severe dengue (16.5%) in our healthcare institution, the sensitivity was 71.4% with a specificity of 87.9% in the febrile phase, and the positive and negative predictive values were 54.7% and 95.8%, respectively. Olfactomedin 4 was elevated in dengue patients but not in proportion to disease severity in the febrile phase (P = .04) There were no significant differences in chymase and tryptase levels between dengue patients and controls. CONCLUSIONS: In adult dengue, suPAR may be a reliable prognostic biomarker for severe dengue in the febrile phase.

Pulmonary function outcomes after tuberculosis treatment in children: a systematic review and meta-analysis.

BACKGROUND: Despite tuberculosis (TB) being a curable disease, current guidelines fail to account for the long-term outcomes of post-tuberculosis lung disease-a cause of global morbidity despite successful completion of effective treatment. Our systematic review aimed to synthesise the available evidence on the lung function outcomes of childhood pulmonary tuberculosis (PTB). METHODS: PubMed, ISI Web of Science, Cochrane Library and ProQuest databases were searched for English-only studies without time restriction (latest search date 22 March 2023). Inclusion criteria were (1) patients who had TB with pulmonary involvement at age ≤18 years; (2) pulmonary function tests (PFTs) performed on patients after treatment completion; and (3) observational studies, including cohort and cross-sectional studies. We adhered to the recommendations of the Cochrane Collaboration and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. RESULTS: From 8040 records, 5 studies were included (involving n=567 children), with spirometry measures from 4 studies included in the meta-analyses. The effect sizes of childhood TB on forced expiratory volume in the first second and forced vital capacity z-scores were estimated to be -1.53 (95% CI -2.65, -0.41; p=0.007) and -1.93 (95% CI -3.35, -0.50; p=0.008), respectively. DISCUSSION: The small number of included studies reflects this under-researched area, relative to the global burden of TB. Nevertheless, as childhood PTB impacts future lung function, PFTs (such as spirometry) should be considered a routine test when evaluating the long-term lung health of children beyond their completion of TB treatment. PROSPERO registration number CRD42021250172.

Rapid Screening of Urinary Tract Infection Using Microfluidic Inertial-Impedance Cytometry.

Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI (n = 20) and UTI patients (n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.

Myeloperoxidase inhibition may protect against endothelial glycocalyx shedding induced by COVID-19 plasma.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19, is a threat to public health. Evidence suggests increased neutrophil activation and endothelial glycocalyx (EG) damage are independently associated with severe COVID-19. Here, we hypothesised that an increased level of blood neutrophil myeloperoxidase (MPO) is associated with soluble EG breakdown, and inhibiting MPO activity may reduce EG damage. METHODS: Analysing a subset of acute and convalescent COVID-19 plasma, 10 from severe and 15 from non-severe COVID-19 cases, and 9 from pre-COVID-19 controls, we determined MPO levels, MPO activity and soluble EG proteins (syndecan-1 and glypican-1) levels by enzyme-linked immunosorbent assay. In vitro primary human aortic endothelial cells were cultured with plasma untreated or treated with specific MPO inhibitors (MPO-IN-28, AZD5904) to determine EG shedding. We then investigated whether inhibiting MPO activity decreased EG degradation. RESULTS: In COVID-19 plasma, MPO levels, MPO activity and levels of soluble EG proteins are significantly raised compared to controls, and concentrations increase in proportion to disease severity. Despite clinical recovery, protein concentrations remain significantly elevated. Interestingly, there is a trend of increasing MPO activity in convalescent plasma in both severe and non-severe groups. MPO levels and MPO activity correlate significantly with soluble EG levels and inhibiting MPO activity leads to reduced syndecan-1 shedding, in vitro. CONCLUSIONS: Neutrophil MPO may increase EG shedding in COVID-19, and inhibiting MPO activity may protect against EG degradation. Further research is needed to evaluate the utility of MPO inhibitors as potential therapeutics against severe COVID-19.

COVID-19 can result in severe disease and is potentially fatal. Neutrophils, the most abundant white blood cells in circulation, secrete antimicrobials that have been linked to severe COVID-19 development. The endothelial glycocalyx (EG) is a carbohydrate rich layer that coats the inner surface of the vasculature and damage to the EG is observed in severe COVID-19. Here, we investigate whether myeloperoxidase, an antimicrobial released by neutrophils, is associated with EG damage in COVID-19 patients. We also determine whether reducing myeloperoxidase activity prevents damage to the EG. Our results suggest myeloperoxidase is associated with EG damage and severe COVID-19. We also demonstrated that a reduction in myeloperoxidase activity may protect against EG degradation. Further studies to evaluate the utility of MPO inhibitors as a therapy against severe COVID-19 are warranted.

An Evidence-Based Serious Game App for Public Education on Antibiotic Use and Antimicrobial Resistance: Protocol of a Randomized Controlled Trial.

BACKGROUND: The misuse and overuse of antibiotics contribute to the acceleration of antimicrobial resistance (AMR), but public knowledge on appropriate antibiotic use and AMR remained low despite ongoing health promotion efforts. App gamification has gained traction in recent years for health promotion and to affect change in health behaviors. Hence, we developed an evidence-based serious game app "SteWARdS Antibiotic Defence" to educate the public on appropriate antibiotic use and AMR and address knowledge gaps. OBJECTIVE: We aim to evaluate the effectiveness of the "SteWARdS Antibiotic Defence" app in improving the knowledge of, attitude toward, and perception (KAP) of appropriate antibiotic use and AMR among the public. The primary objective is to assess the changes in KAP of antibiotic use and AMR in our participants, while the secondary objectives are to assess the extent of user engagement with the app and the level of user satisfaction in using the app. METHODS: Our study is a parallel 2-armed randomized controlled trial with a 1:1 allocation. We plan to recruit 400 participants (patients or their caregivers) aged 18-65 years from government-funded primary care clinics in Singapore. Participants are randomized in blocks of 4 and into the intervention or control group. Participants in the intervention group are required to download the "SteWARdS Antibiotic Defence" app on their smartphones and complete a game quest within 2 weeks. Users will learn about appropriate antibiotic use and effective methods to recover from uncomplicated upper respiratory tract infections by interacting with the nonplayer characters and playing 3 minigames in the app. The control group will not receive any intervention. RESULTS: The primary study outcome is the change in participants' KAP toward antibiotic use and AMR 6-10 weeks post intervention or 6-10 weeks from baseline for the control group (web-based survey). We will also assess the knowledge level of participants immediately after the participant completes the game quest (in the app). The secondary study outcomes are the user engagement level (tracked by the app) and satisfaction level of playing the game (via the immediate postgame survey). The satisfaction survey will also collect participants' feedback on the game app. CONCLUSIONS: Our proposed study provides a unique opportunity to assess the effectiveness of a serious game app in public health education. We anticipate possible ceiling effects and selection bias in our study and have planned to perform subgroup analyses to adjust for confounding factors. The app intervention will benefit a larger population if it is proven to be effective and acceptable to users. TRIAL REGISTRATION: ClinicalTrials.gov NCT05445414; https://clinicaltrials.gov/ct2/show/NCT05445414. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/45833.

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

DYNAMIC cohort study evaluating metabolic predictors of influenza vaccine immune response in older adults.

Immunosenescence (age-related immune dysfunction) and inflamm-aging contribute to suboptimal immune responses in older adults to standard-dose influenza vaccines, which may be exacerbated in those with metabolic co-morbidities. We sought to investigate metabolic factors/predictors of influenza vaccine immune response in an older adult (age ≥65 years) cohort in Singapore, where influenza typically circulates year-round. The primary outcome for the DYNAMIC prospective cohort study was haemagglutination-inhibition titer (HAI) response to each of the trivalent inactivated influenza vaccine strains at day 28 (D28) compared to baseline (D0), as assessed by seroconversion and D28/D0 log2 HAI fold rise. Baseline blood samples were tested for total Vitamin D (25-(OH) D) levels. We enrolled 234 participants in June-Dec 2017. Two hundred twenty completed all study visits. The median age was 71 [IQR 68-75] years, 67 (30.5%) had diabetes mellitus (DM), and the median BMI was 24.9 [IQR 22.2-27.8] kg/m2. Median baseline totals 25-(OH) D was 29 [IQR: 21-29] ng/ml. Age, DM, obesity, and baseline 25-(OH) D were not associated with HAI fold rise in multivariable analysis. More recent prior influenza vaccination and higher baseline HAI titers were associated with lower HAI fold rise for influenza A/HK/H3N2. Physical activity was associated with a higher HAI fold rise for influenza A/HK/H3N2 in a dose-response relationship (p-test for trend = 0.015). Older adults with well-controlled metabolic co-morbidities retain HAI response to the influenza vaccine, and physical activity had a beneficial effect on immune response, particularly for influenza A/HK/H3N2.

Clinical and prognostic relevance of sST2 in adults with dengue-associated cardiac impairment and severe dengue.

BACKGROUND: Dengue can be complicated by severe outcomes including cardiac impairment, and the lack of reliable prognostic biomarkers poses a challenge in managing febrile dengue patients. Here, we investigated the functionality of soluble suppressor of tumorigenicity (sST2) as a predictive marker of severe dengue and its association in dengue-associated cardiac impairment. METHODS: Plasma samples, aged >16 years, collected from 36 dengue fever, 43 dengue with warning signs, 11 severe dengue (collected at febrile, critical and recovery phases) and 30 controls were assayed for plasma levels of sST2, troponin T and N-terminal (NT)-pro hormone brain natriuretic peptide (NT-proBNP) by ELISA. Cardiac parameters: stroke index (SI), cardiac index (CI) and Granov-Goor Index (GGI) were measured with a bioimpedance device during the different phases for dengue subjects and once for the controls. PRINCIPAL FINDINGS: In the febrile, critical and early recovery phases, sST2 levels were significantly elevated in dengue participants and sST2 levels increased with increasing disease severity (P < 0.01 for all). sST2 concentrations were negatively correlated with SI (r = -0.48; P < 0.001, r = -0.55; P < 0.001), CI (r = -0.26; P = 0.02, r = -0.6: P < 0.001) and GGI (r = -0.44; P < 0.001, r = -0.57; P < 0.001) in the critical and early recovery phases. In contrast, sST2 levels in the febrile and critical phases, were positive correlated to troponin T (r = 0.44, P < 0.001; r = 0.22, P = 0.03, respectively) and NT-proBNP (r = 0.21, P = 0.03; r = 0.35, P < 0.001). ROC analysis demonstrated sST2 as a good biomarker of severe dengue in the critical phase, AUROC 0.79, P < 0.001. CONCLUSION/SIGNIFICANCE: sST2 levels were elevated in patients with dengue especially in cases of severe dengue. Furthermore, increased sST2 levels were associated with cardiac indicators suggesting lower cardiac performance. While further research is needed to demonstrate its clinical utility, sST2 may be a useful prognostic biomarker of severe dengue.

Association of Neutrophil Mediators With Dengue Disease Severity and Cardiac Impairment in Adults.

BACKGROUND: Cardiac impairment contributes to hypotension in severe dengue (SD). However, studies examining pathogenic factors affecting dengue-associated cardiac impairment are lacking. We examined the role of neutrophil mediators on cardiac impairment in clinical dengue. METHODS: We prospectively enrolled adult patients with dengue and controls. Cardiac parameters were measured using a bioimpedance device. Neutrophils mediators were measured, including myeloperoxidase (MPO) and citrullinated histone H3. RESULTS: We recruited 107 dengue patients and 30 controls. Patients with dengue were classified according to World Health Organization 2009 guidelines (44 with dengue fever [DF], 51 with DF with warning signs, and 12 with SD). During critical phase, stroke index (P < .001), cardiac index (P = .03), and Granov-Goor index (P < .001) were significantly lower in patients with dengue than in controls. During critical phase, MPO was significantly higher in patients with dengue than in controls (P < .001) and also significantly higher in patients with SD than in those with DF. In addition, MPO was inversely associated with the stroke, cardiac, and Granov-Goor indexes, during the critical phase, and longitudinally as well. CONCLUSIONS: Cardiac function was decreased, and MPO increased, during with critical phase in patients SD compared with those with DF and controls. MPO may mediate dengue-associated cardiac impairment.

A double whammy: The association between comorbidities and severe dengue among adult patients-A matched case-control study.

BACKGROUND: Dengue infection is the most prevalent mosquito-borne viral infection globally. Concurrently, there has also been an upsurge of non-communicable comorbidities. We aimed to investigate the association between these comorbidities and the development of severe dengue. METHODS: We performed a retrospective, case-control study involving 117 cases with severe dengue and 351 controls with non-severe dengue; matched according to gender, age (+/- 5 years old), and admission date (+/- 2 weeks). We analyzed the data using conditional odds ratio (cOR) and adjusted conditional odds ratio (AcOR) using univariate and multivariable conditional logistic regression respectively. RESULTS: Six main comorbidities namely obesity, diabetes mellitus, hypertension, hyperlipidemia, chronic pulmonary disease, and ischemic heart disease were observed among cases and controls. Multivariable conditional logistic regression model found only hypertension to be independently associated with the development of severe dengue (ACOR 2.46; 95% CI:1.09-5.53). Among symptoms at presentation, lethargy, vomiting, bleeding manifestations, and abdominal pain were associated with increased odds of severe dengue, although the associations were not statistically significant. Headache (ACOR: 0:32; 95% CI: 0.21-0.51) and skin rash (ACOR: 0.42; 95% CI: 0.22-0.81) were associated with significantly lower odds of severe dengue. Severe dengue patients were also found to have significantly higher white cell count, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase on admission, while platelet and albumin were significantly lower compared to non-severe dengue patients. CONCLUSIONS: Our study found a significant association between hypertension and the development of severe dengue in adult patients. For clinical practice, this finding suggests that dengue patients with underlying hypertension warrant closer clinical monitoring for deterioration. The association between significant derangement in various laboratory parameters and severe dengue as shown in this study is in keeping with previous reports. While further substantiation by larger prospective studies will be desirable, this association may serve to inform the dengue triaging process.

Association of systemic vitamin D on the course of dengue virus infection in adults: a single-centre dengue cohort study at a large institution in Singapore.

INTRODUCTION: Host immune responses may impact dengue severity in adults. Vitamin D has multiple immunomodulatory effects on innate and adaptive immunity. METHODS: We evaluated the association between systemic 25-hydroxyvitamin D [25-(OH) D] and dengue disease severity in adults. We measured plasma for total 25-(OH) D levels with an electrochemiluminescence immunoassay using stored samples from participants with laboratory confirmed dengue who were prospectively enrolled in 2012-2016 at our institution. RESULTS: 80 participants (median age 43 years) were enrolled. Six participants had severe dengue based on the World Health Organisation (WHO) 1997 criteria (i.e. dengue haemorrhagic fever/dengue shock syndrome) and another six had severe dengue based on the WHO 2009 criteria. Median 25-(OH) D at acute phase of dengue was 6.175 µg/L (interquartile range 3.82-8.21; range 3.00-15.29) in all participants. 25-(OH) D showed inverse linear trend with severe dengue manifestations based on the WHO 2009 criteria (aRR 0.72; 95% confidence interval 0.57-0.91; p < 0.01) after adjustment for age, gender and ethnicity. CONCLUSION: Limited studies have evaluated the role of systemic 25-(OH) D on dengue severity. Our study found low systemic 25-(OH) D was associated with increased dengue disease severity, particularly for severe bleeding that was not explained by thrombocytopenia. Further studies investigating the underlying immune mechanisms and effects on the vascular endothelium are needed.

Malaria in Pregnancy: From Placental Infection to Its Abnormal Development and Damage.

Malaria remains a global health burden with Plasmodium falciparum accounting for the highest mortality and morbidity. Malaria in pregnancy can lead to the development of placental malaria, where P. falciparum-infected erythrocytes adhere to placental receptors, triggering placental inflammation and subsequent damage, causing harm to both mother and her infant. Histopathological studies of P. falciparum-infected placentas revealed various placental abnormalities such as excessive perivillous fibrinoid deposits, breakdown of syncytiotrophoblast integrity, trophoblast basal lamina thickening, increased syncytial knotting, and accumulation of mononuclear immune cells within intervillous spaces. These events in turn, are likely to impair placental development and function, ultimately causing placental insufficiency, intrauterine growth restriction, preterm delivery and low birth weight. Hence, a better understanding of the mechanisms behind placental alterations and damage during placental malaria is needed for the design of effective interventions. In this review, using evidence from human studies and murine models, an integrated view on the potential mechanisms underlying placental pathologies in malaria in pregnancy is provided. The molecular, immunological and metabolic changes in infected placentas that reflect their responses to the parasitic infection and injury are discussed. Finally, potential models that can be used by researchers to improve our understanding on the pathogenesis of malaria in pregnancy and placental pathologies are presented.

Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar.

Multidrug resistance is a major threat to global elimination of tuberculosis (TB). We performed phenotypic drug-susceptibility testing and whole-genome sequencing for 309 isolates from 342 consecutive patients who were given a diagnosis of TB in Yangon, Myanmar, during July 2016‒June 2018. We identified isolates by using the GeneXpert platform to evaluate drug-resistance profiles. A total of 191 (62%) of 309 isolates had rifampin resistance; 168 (88%) of these rifampin-resistant isolates were not genomically related, indicating the repeated emergence of resistance in the population, rather than extensive local transmission. We did not detect resistance mutations to new oral drugs, including bedaquiline and pretomanid. The current GeneXpert MTB/RIF system needs to be modified by using the newly launched Xpert MTB/XDR cartridge or line-probe assay. Introducing new oral drugs to replace those currently used in treatment regimens for multidrug-resistant TB will also be useful for treating TB in Myanmar.

Association between lung compliance phenotypes and mortality in COVID-19 patients with acute respiratory distress syndrome.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) in COVID-19 is associated with a high mortality rate, though outcomes of the different lung compliance phenotypes are unclear. We aimed to measure lung compliance and examine other factors associated with mortality in COVID-19 patients with ARDS. METHODS: Adult patients with COVID-19 ARDS who required invasive mechanical ventilation at 8 hospitals in Singapore were prospectively enrolled. Factors associated with both mortality and differences between high (<40mL/cm H2O) and low (<40mL/cm H2O) compliance were analysed. RESULTS: A total of 102 patients with COVID-19 who required invasive mechanical ventilation were analysed; 15 (14.7%) did not survive. Non-survivors were older (median 70 years, interquartile range [IQR] 67-75 versus median 61 years, IQR 52-66; P<0.01), and required a longer duration of ventilation (26 days, IQR 12-27 vs 8 days, IQR 5-15; P<0.01) and intensive care unit support (26 days, IQR 11-30 vs 11.5 days, IQR 7-17.3; P=0.01), with a higher incidence of acute kidney injury (15 patients [100%] vs 40 patients [46%]; P<0.01). There were 67 patients who had lung compliance data; 24 (35.8%) were classified as having high compliance and 43 (64.2%) as having low compliance. Mortality was higher in patients with high compliance (33.3% vs 11.6%; P=0.03), and was associated with a drop in compliance at day 7 (-9.3mL/cm H2O (IQR -4.5 to -15.4) vs 0.2mL/cm H2O (4.7 to -5.2) P=0.04). CONCLUSION: COVID-19 ARDS patients with higher compliance on the day of intubation and a longitudinal decrease over time had a higher risk of death.

Tracking oxidation-induced alterations in fibrin clot formation by NMR-based methods.

Plasma fibrinogen is an important coagulation factor and susceptible to post-translational modification by oxidants. We have reported impairment of fibrin polymerization after exposure to hypochlorous acid (HOCl) and increased methionine oxidation of fibrinogen in severely injured trauma patients. Molecular dynamics suggests that methionine oxidation poses a mechanistic link between oxidative stress and coagulation through protofibril lateral aggregation by disruption of AαC domain structures. However, experimental evidence explaining how HOCl oxidation impairs fibrinogen structure and function has not been demonstrated. We utilized polymerization studies and two dimensional-nuclear magnetic resonance spectrometry (2D-NMR) to investigate the hypothesis that HOCl oxidation alters fibrinogen conformation and T2 relaxation time of water protons in the fibrin gels. We have demonstrated that both HOCl oxidation of purified fibrinogen and addition of HOCl-oxidized fibrinogen to plasma fibrinogen solution disrupted lateral aggregation of protofibrils similarly to competitive inhibition of fibrin polymerization using a recombinant AαC fragment (AαC 419-502). DOSY NMR measurement of fibrinogen protons demonstrated that the diffusion coefficient of fibrinogen increased by 17.4%, suggesting the oxidized fibrinogen was more compact and fast motion in the prefibrillar state. 2D-NMR analysis reflected that water protons existed as bulk water (T2) and intermediate water (T2i) in the control plasma fibrin. Bulk water T2 relaxation time was increased twofold and correlated positively with the level of HOCl oxidation. However, T2 relaxation of the oxidized plasma fibrin gels was dominated by intermediate water. Oxidation induced thinner fibers, in which less water is released into the bulk and water fraction in the hydration shell was increased. We have confirmed that T2 relaxation is affected by the self-assembly of fibers and stiffness of the plasma fibrin gel. We propose that water protons can serve as an NMR signature to probe oxidative rearrangement of the fibrin clot.

Immune cell phenotypes associated with disease severity and long-term neutralizing antibody titers after natural dengue virus infection.

Prior immunological exposure to dengue virus can be both protective and disease-enhancing during subsequent infections with different dengue virus serotypes. We provide here a systematic, longitudinal analysis of B cell, T cell, and antibody responses in the same patients. Antibody responses as well as T and B cell activation differentiate primary from secondary responses. Hospitalization is associated with lower frequencies of activated, terminally differentiated T cells and higher percentages of effector memory CD4 T cells. Patients with more severe disease tend to have higher percentages of plasmablasts. This does not translate into long-term antibody titers, since neutralizing titers after 6 months correlate with percentages of specific memory B cells, but not with acute plasmablast activation. Overall, our unbiased analysis reveals associations between cellular profiles and disease severity, opening opportunities to study immunopathology in dengue disease and the potential predictive value of these parameters.