Search

The COVID-19 pandemic has been influencing people’s psychological health, especially in pregnant women. We aimed to examine associated factors of fear of COVID-19, anxiety and depression among pregnant women during the pandemic where the impacts of healthy eating behaviour (HES) and health literacy (HL) were emphasized. A cross-sectional study was conducted between 14 February 2020 and 31 May 2020 in 18 health centres and hospitals across Vietnam. Data of 518 pregnant women were analysed, including socio-demographics, pregnant-related factors, HES, HL, health-related behaviours, fear of COVID-19 scale (FCoV-19S), anxiety (using the generalized anxiety disorder (GAD-7)) and depression (using the patient health questionnaire with 9 items (PHQ-9)). Regression analysis was utilized to explore the associations. Pregnant women with higher scores of HES and HL had lower likelihood of anxiety (odds ratio, OR, 0.79; 95% confidence interval (95%CI), 0.73, 0.87; p < .001; and OR, 0.94; 95%CI, 0.90, 0.99; p = .018) and depression (OR, 0.84; 95%CI, 0.78, 0.91; p < .001; and OR, 0.96; 95%CI, 0.91, 0.99; p = .044), respectively. Pregnant women being employed had a lower FCoV-19S score (regression coefficient, B, −1.46; 95%CI, −2.51, −0.40; p = .007). Besides, other significant predictors of anxiety were eating healthier during the pandemic, unchanged or more physical activity, elevated gestational age and smoking. Other significant predictors of depression were eating healthier during the pandemic, elevated gestational age and smoking. Among others, HES and HL had positive impacts on protecting pregnant women against anxiety and depression. Improving HES and HL should be addressed as a strategic approach to improve reproductive health during the pandemic.KEY MESSAGEThe COVID-19 pandemic influences antenatal mental disorders with the higher level as opposed to that before the pandemic.Healthy eating behaviour and better health literacy (HL) had critical roles in lowering prenatal anxiety and depression during the COVID-19 crisis.Strategic approaches for improving healthy eating and HL should be recommended for protecting pregnant women from mental health problems during the pandemic. The COVID-19 pandemic influences antenatal mental disorders with the higher level as opposed to that before the pandemic. Healthy eating behaviour and better health literacy (HL) had critical roles in lowering prenatal anxiety and depression during the COVID-19 crisis. Strategic approaches for improving healthy eating and HL should be recommended for protecting pregnant women from mental health problems during the pandemic.

ObjectivesWe examined impacts and interactions of COVID-19 response involvement, health-related behaviours and health literacy (HL) on anxiety, depression, and health-related quality of life (HRQoL) among healthcare workers (HCWs).DesignA cross-sectional study was conducted. Data were collected 6 April to 19 April 2020 using online-based, self-administered questionnaires.Setting19 hospitals and health centres in Vietnam.Participants7 124 HCWs aged 21–60 years.ResultsThe COVID-19 response-involved HCWs had higher anxiety likelihood (OR (95% CI)=4.41 (3.53 to 5.51)), higher depression likelihood (OR(95% CI)=3.31 (2.71 to 4.05)) and lower HRQoL score (coefficient, b(95% CI)=−2.14 (−2.89 to −1.38)), compared with uninvolved HCWs. Overall, HCWs who smoked or drank at unchanged/increased levels had higher likelihood of anxiety, depression and lower HRQoL scores; those with unchanged/healthier eating, unchanged/more physical activity and higher HL scores had lower likelihood of anxiety, depression and higher HRQoL scores. In comparison to uninvolved HCWs who smoked or drank at never/stopped/reduced levels, involved HCWs with unchanged/increased smoking or drinking had lower anxiety likelihood (OR(95% CI)=0.34 (0.14 to 0.83)) or (OR(95% CI)=0.26 (0.11 to 0.60)), and lower depression likelihood (OR(95% CI)=0.33 (0.15 to 0.74)) or (OR(95% CI)=0.24 (0.11 to 0.53)), respectively. In comparison with uninvolved HCWs who exercised at never/stopped/reduced levels, or with those in the lowest HL quartile, involved HCWs with unchanged/increased exercise or with one-quartile HL increment reported lower anxiety likelihood (OR(95% CI)=0.50 (0.31 to 0.81)) or (OR(95% CI)=0.57 (0.45 to 0.71)), lower depression likelihood (OR(95% CI)=0.40 (0.27 to 0.61)) or (OR(95% CI)=0.63 (0.52 to 0.76)), and higher HRQoL scores (b(95% CI)=2.08 (0.58 to 3.58)), or (b(95% CI)=1.10 (0.42 to 1.78)), respectively.ConclusionsPhysical activity and higher HL were found to protect against anxiety and depression and were associated with higher HRQoL. Unexpectedly, smoking and drinking were also found to be coping behaviours. It is important to have strategic approaches that protect HCWs’ mental health and HRQoL.

Background The COVID-19 pandemic has imposed a heavy burden on health care systems and governments. Health literacy (HL) and eHealth literacy (as measured by the eHealth Literacy Scale [eHEALS]) are recognized as strategic public health elements but they have been underestimated during the pandemic. HL, eHEALS score, practices, lifestyles, and the health status of health care workers (HCWs) play crucial roles in containing the COVID-19 pandemic. Objective The aim of this study is to evaluate the psychometric properties of the eHEALS and examine associations of HL and eHEALS scores with adherence to infection prevention and control (IPC) procedures, lifestyle changes, and suspected COVID-19 symptoms among HCWs during lockdown. Methods We conducted an online survey of 5209 HCWs from 15 hospitals and health centers across Vietnam from April 6 to April 19, 2020. Participants answered questions related to sociodemographics, HL, eHEALS, adherence to IPC procedures, behavior changes in eating, smoking, drinking, and physical activity, and suspected COVID-19 symptoms. Principal component analysis, correlation analysis, and bivariate and multivariate linear and logistic regression models were used to validate the eHEALS and examine associations. Results The eHEALS had a satisfactory construct validity with 8 items highly loaded on one component, with factor loadings ranked from 0.78 to 0.92 explaining 76.34% of variance; satisfactory criterion validity as correlated with HL (ρ=0.42); satisfactory convergent validity with high item-scale correlations (ρ=0.80-0.84); and high internal consistency (Cronbach α=.95). HL and eHEALS scores were significantly higher in men (unstandardized coefficient [B]=1.01, 95% CI 0.57-1.45, P Conclusions The eHEALS is a valid and reliable survey tool. Gender, ability to pay for medication, profession, and epidemic containment experience were independent predictors of HL and eHEALS scores. HCWs with higher HL or eHEALS scores had better adherence to IPC procedures, healthier lifestyles, and a lower likelihood of suspected COVID-19 symptoms. Efforts to improve HCWs’ HL and eHEALS scores can help to contain the COVID-19 pandemic and minimize its consequences.

BACKGROUND The COVID-19 pandemic has imposed a heavy burden on health care systems and governments. Health literacy (HL) and eHealth literacy (as measured by the eHealth Literacy Scale [eHEALS]) are recognized as strategic public health elements but they have been underestimated during the pandemic. HL, eHEALS score, practices, lifestyles, and the health status of health care workers (HCWs) play crucial roles in containing the COVID-19 pandemic. OBJECTIVE The aim of this study is to evaluate the psychometric properties of the eHEALS and examine associations of HL and eHEALS scores with adherence to infection prevention and control (IPC) procedures, lifestyle changes, and suspected COVID-19 symptoms among HCWs during lockdown. METHODS We conducted an online survey of 5209 HCWs from 15 hospitals and health centers across Vietnam from April 6 to April 19, 2020. Participants answered questions related to sociodemographics, HL, eHEALS, adherence to IPC procedures, behavior changes in eating, smoking, drinking, and physical activity, and suspected COVID-19 symptoms. Principal component analysis, correlation analysis, and bivariate and multivariate linear and logistic regression models were used to validate the eHEALS and examine associations. RESULTS The eHEALS had a satisfactory construct validity with 8 items highly loaded on one component, with factor loadings ranked from 0.78 to 0.92 explaining 76.34% of variance; satisfactory criterion validity as correlated with HL (ρ=0.42); satisfactory convergent validity with high item-scale correlations (ρ=0.80-0.84); and high internal consistency (Cronbach α=.95). HL and eHEALS scores were significantly higher in men (unstandardized coefficient [B]=1.01, 95% CI 0.57-1.45, PPPPPP=.003), and those with epidemic containment experience (B=1.96, 95% CI 1.56-2.37, PPPPP=.001; OR 1.04, 95% CI 1.02-1.07, P=.002), were more physically active (OR 1.03, 95% CI 1.02-1.03, PPPP CONCLUSIONS The eHEALS is a valid and reliable survey tool. Gender, ability to pay for medication, profession, and epidemic containment experience were independent predictors of HL and eHEALS scores. HCWs with higher HL or eHEALS scores had better adherence to IPC procedures, healthier lifestyles, and a lower likelihood of suspected COVID-19 symptoms. Efforts to improve HCWs’ HL and eHEALS scores can help to contain the COVID-19 pandemic and minimize its consequences.

The coronavirus disease 2019 (COVID-19) epidemic affects people&rsquo, s health and health-related quality of life (HRQoL), especially in those who have suspected COVID-19 symptoms (S-COVID-19-S). We examined the effect of modifications of health literacy (HL) on depression and HRQoL. A cross-sectional study was conducted from 14 February to 2 March 2020. 3947 participants were recruited from outpatient departments of nine hospitals and health centers across Vietnam. The interviews were conducted using printed questionnaires including participants&rsquo, characteristics, clinical parameters, health behaviors, HL, depression, and HRQoL. People with S-COVID-19-S had a higher depression likelihood (OR, 2.88, p &lt, 0.001), lower HRQoL-score (B, &minus, 7.92, 0.001). In comparison to people without S-COVID-19-S and low HL, those with S-COVID-19-S and low HL had 9.70 times higher depression likelihood (p &lt, 0.001), 20.62 lower HRQoL-score (p &lt, 0.001), for the people without S-COVID-19-S, 1 score increment of HL resulted in 5% lower depression likelihood (p &lt, 0.001) and 0.45 higher HRQoL-score (p &lt, 0.001), while for those people with S-COVID-19-S, 1 score increment of HL resulted in a 4% lower depression likelihood (p = 0.004) and 0.43 higher HRQoL-score (p &lt, 0.001). People with S-COVID-19-S had a higher depression likelihood and lower HRQoL than those without. HL shows a protective effect on depression and HRQoL during the epidemic.

Numerous studies point to the utility of blood cytokine measurements in the diagnosis and treatment of human disease. Advances in detection allow robust multiplex analysis. However, cytokines are present at low levels and are produced and act in complex networks which can remain active in stored blood. A major barrier to the routine use of cytokines as clinical biomarkers is sample management prior to analysis. Studies on cytokine stability under storage frequently use ‘spiked’ normal control plasma or serum to generate detectable levels of the cytokines of interest. These conditions may oversimplify the reality of clinically complex samples and provide limited information regarding optimal management of whole blood samples prior to plasma separation. Cytokine stability has also been addressed previously using plasma from normal individuals under different conditions of anticoagulant use, storage time and temperature of storage. No studies have as yet been undertaken to address cytokine stability in critically ill patients which may differ from normal, healthy individuals due to underlying cofounders such as inflammation. To address these issues, we subjected samples from five patients exhibiting an inflammatory disease state to three storage extremes which might be encountered in a clinical setting, prior to analysis of 40 cytokines. Blood drawn into EDTA or ACD anticoagulant was immediately separated and plasma stored at −80 °C. Matched samples were stored as follows; whole blood overnight at room temperature, or whole blood or plasma stored 10 days at 4 °C. We used equivalence testing to determine the similarity of stored cytokine values to baseline values. In ACD plasma, Eotaxin, IL-6, IL-11, IL-15, IP10, MDC, MCP-1 met equivalence to baseline in all storage conditions while for EDTA plasma stored 10 days at 4 °C EGF, FGF2, Fractalkine, G-CSF, IL-1β, IL-5, IL-6, IL-7, IL-11, IP-10, TGFα and TNFα showed equivalence to baseline measurements. Intraclass Correlation Coefficients (ICC) were calculated and the following cytokines showed good to excellent agreement across all 4 storage conditions: Eotaxin, IL-5, IL-6, IL-11, IL-13, IP-10, MCP-1 and TNFα when collected in EDTA, and Eotaxin, IL-5, IL-6, IL-11, IL12-p40, IL-15, IP-10 and MCP-1 when collected in ACD. Five plasma cytokines were identified as being the least stable in both ACD and EDTA: IL-7, IL-9, IL12p70, RANTES, sCD40L, while IL-1β was identified as unstable stored in ACD plasma. This study identified several clinically important cytokines that are remarkably stable in blood and plasma, and some that stored poorly. To our knowledge, this is the first cytokine storage study to use medically unwell patient samples and equivalence testing to evaluate the stability of measured cytokine values after storage.

People with pre-existing health conditions (PEHC) are vulnerable to viral infection while health literacy (HL) and preventive behaviors (PB) have been shown to benefit people during the COVID-19 pandemic. The aim of this study was to examine the association between PEHC and suspected COVID-19 symptoms (S-COVID-19-S), and to investigate the modification effect of HL and PB. A cross-sectional study was conducted on 8291 participants visiting outpatient departments at 18 hospitals and health centers across Vietnam from 14 February to 31 May 2020. Data were collected regarding participant&rsquo, s characteristics, HL, PB, PEHC, and S-COVID-19-S. Regression models were used for analyzing the associations. Results showed that people with PEHC had a 3.38 times higher likelihood of having S-COVID-19-S (odds ratio, OR, 3.38, 95% confidence interval, 95% CI, 3.01, 3.79, p &lt, 0.001). In comparison to participants without PEHC and with the lowest HL score, those with PEHC and one HL score increment had a 7% lower likelihood of having S-COVID-19-S (OR, 0.93, 95% CI, 0.92, 0.94, 0.001). In comparison to participants without PEHC and not adhering to mask wearing, those with PEHC and adhering to mask wearing had a 77% lower likelihood of having S-COVID-19-S (OR, 0.23, 95% CI, 0.16, 0.32, 0.001). Higher HL and adherence to mask wearing can protect people from having S-COVID-19-S, especially in those with PEHC.

Background: Heterogeneity in response to biological medication is a major challenge in the management of Juvenile Idiopathic Arthritis (JIA) (1). Biomarkers such as cytokines or whole blood RNA expression profiles that predict therapy efficacy prior to treatment pose a solution and could enable precision medicine. It was previously demonstrated that whole blood gene expression profiles can predict response to anti-TNF-α therapy in patients with JIA (2). These findings currently await validation in a multicenter prospective cohort study. Harmonization of data from different cohorts, however, is often hampered by site-dependent differences in biological sample collection and processing (3). For instance, the biological material that is retrospectively available from biobanks depends on what is stored exactly, e.g. whole blood versus peripheral blood mononuclear cells (PBMC). Furthermore, different blood collection tubes containing different additives can affect sample quality at an early stage and make interchangeability or comparability of data impossible. Objectives: We evaluated the comparability of gene expression profiles when whole blood from patients with JIA is collected in different RNA collection tubes and processed accordingly. Next, we will investigate the predictive capacity for therapy response of RNA expression profiles from frozen PBMC of 63 JIA patients. Methods: Peripheral blood from 11 children with non-systemic JIA with active disease was collected in PAXgene (PreAnalytiX) and Tempus (Applied Biosystems) tubes. All tubes were subsequently stored in the freezer at -80 °C. After thawing, RNA from the PAXgene tubes was extracted with use of the PAXgene Blood RNA Kit and RNA from the Tempus tubes was isolated by using the Tempus Preserved Blood RNA Purification Kit I. Gene expression of CSNK1D, C1D, ASAP2, SRRD, PPP1R3B, HLA-DQA1, PDZK1IP1 and MZB1 was determined with qPCR. For our next experiment, frozen PBMC derived from patients with non-systemic JIA who are included in our longitudinal Pharmachild registry biobank will be used. Of the 63 patients, 28 (44.4%) children were diagnosed with oligo articular JIA, 19 (30.2%) with poly articular JIA, 9 (14.3%) with enthesitis-related JIA, 4 (6.3%) with psoriatic JIA and 3 (4.8%) with undifferentiated JIA. The average age at sampling was 11.5 years ± 4.0 and 65% of the patients was female. These samples will be run on a 96-analyte NanoString panel and associated with clinical response at time points 3 and 6 months after start of TNF-α blockade. Results: Both RNA blood collection systems yielded high-quality RNA, with overall higher RNA concentrations for blood collected in Tempus tubes in comparison to PAXgene tubes. qPCR data showed that gene expression of all measured genes is affected by the method of blood collection and processing. However, the inter-individual variation was similar between both collection tubes, indicating that similar RNA profiles were observed. Conclusion: Gene expression profiles derived from whole blood collected in PAXgene or Tempus tubes are comparable, but are not interchangeable. References: [1] Swart JF, de Roock S, & Prakken BJ. Understanding inflammation in juvenile idiopathic arthritis: How immune biomarkers guide clinical strategies in the systemic onset subtype. Eur J Immunol 2016;46(9):2068-2077. [2] Cui A, Quon G, Rosenberg AM et al. Gene Expression Deconvolution for Uncovering Molecular Signatures in Response to Therapy in Juvenile Idiopathic Arthritis. PloS one 2016;11(5):1-17. [3] Yeung RS, Albani S, Feldman BM et al. Enhancing translational research in paediatric rheumatology through standardization. Nature Reviews Rheumatology 2016;12(11):684. Disclosure of Interests: None declared

Background: Healthy eating and physical activity are effective non-pharmacological approaches to boost immune function and contain the pandemic. We aimed to explore the associations and interactions between physical activity and healthy eating behavior with COVID-19-like symptoms (Slike-CV19S). Methods: A cross-sectional study was conducted on 3947 outpatients, from 14 February to 2 March 2020, at nine health facilities in Vietnam. Data collection included sociodemographic characteristics, healthy eating behavior (using the healthy eating score (HES) questionnaire), physical activity (using the short form international physical activity questionnaire), and Slike-CV19S. The associations and interactions were tested using logistic regression models. Results: Frequent intake of fruits (OR = 0.84, p = 0.016), vegetables (OR = 0.72, p = 0.036), and fish (OR = 0.43, p &lt, 0.001) were associated with a lower Slike-CV19S likelihood, as compared with infrequent intake. Patients with higher HES levels (OR = 0.84, p = 0.033 for medium HES, OR = 0.77, p = 0.006 for high HES) or being physically active (OR = 0.69, 0.001) had a lower Slike-CV19S likelihood, as compared to those with low HES or physical inactivity, respectively. Patients with medium HES who were physically active (OR = 0.69, p = 0.005), or with high HES and physically active (OR = 0.58, 0.001), had a lower Slike-CV19S likelihood, as compared to those with low HES and physical inactivity. Conclusions: Healthy eating behavior and physical activity showed single and combinative impacts on protecting people from Slike-CV19S. Strategic approaches are encouraged to improve healthy behaviors, which may further contribute to containing the pandemic.

Background: We aimed to examine the impacts of digital healthy diet literacy (DDL) and healthy eating behaviors (HES) on fear of COVID-19, changes in mental health, and health-related quality of life (HRQoL) among front-line healthcare workers (HCWs). Methods: An online survey was conducted at 15 hospitals and health centers from 6–19 April 2020. Data of 2299 front-line HCWs were analyzed—including socio-demographics, symptoms like COVID-19, health literacy, eHealth literacy, DDL, HES, fear of COVID-19, changes in mental health, and HRQoL. Regression models were used to examine the associations. Results: HCWs with higher scores of DDL and HES had lower scores of FCoV-19S (regression coefficient, B, −0.04; 95% confidence interval, 95% CI, −0.07, −0.02; p = 0.001; and B, −0.10; 95% CI, −0.15, −0.06; p < 0.001); had a higher likelihood of stable or better mental health status (odds ratio, OR, 1.02; 95% CI, 1.00, 1.05; p = 0.029; and OR, 1.04; 95% CI, 1.00, 1.07; p = 0.043); and HRQoL (OR, 1.02; 95% CI, 1.01, 1.03; p = 0.006; and OR, 1.04; 95% CI, 1.02, 1.06; p = 0.001), respectively. Conclusions: DDL and HES were found as independent predictors of fear of COVID−19, changes in mental health status, and HRQoL in front-line HCWs. Improving DDL and HES should be considered as a strategic approach for hospitals and healthcare systems.

Kawasaki disease (KD) is a multisystem vasculitis that predominantly targets the coronary arteries in children. Phenotypic similarities between KD and recurrent fever syndromes point to the potential role of inflammasome activation in KD. Mutations in NLRP3 are associated with recurrent fever/autoinflammatory syndromes. We show that the KD-associated genetic polymorphism in inositol-triphosphate 3-kinase C (ITPKC) (rs28493229) has important functional consequences, governing ITPKC protein levels and thereby intracellular calcium, which in turn regulates NLRP3 expression and production of IL-1β and IL-18. Analysis of transcript abundance, protein levels, and cellular response profiles from matched, serial biospecimens from a cohort of genotyped KD subjects points to the critical role of ITPKC in mediating NLRP3 inflammasome activation. Treatment failure in those with the high-risk ITPKC genotype was associated with the highest basal and stimulated intracellular calcium levels and with increased cellular production of IL-1β and IL-18 and higher circulating levels of both cytokines. Mechanistic studies using Itpkc-deficient mice in a disease model support the genomic, cellular, and clinical findings in affected children. Our findings provide the mechanism behind the observed efficacy of rescue therapy with IL-1 blockade in recalcitrant KD, and we identify that regulation of calcium mobilization is fundamental to the underlying immunobiology in KD.

Objective Childhood arthritis encompasses a heterogeneous family of diseases. Significant variation in clinical presentation remains despite consensus-driven diagnostic classifications. Developments in data analysis provide powerful tools for interrogating large heterogeneous data sets. We report a novel approach to integrating biologic and clinical data toward a new classification for childhood arthritis, using computational biology for data-driven pattern recognition. Methods Probabilistic principal components analysis was used to transform a large set of data into 4 interpretable indicators or composite variables on which patients were grouped by cluster analysis. Sensitivity analysis was conducted to determine key variables in determining indicators and cluster assignment. Results were validated against an independent validation cohort. Results Meaningful biologic and clinical characteristics, including levels of proinflammatory cytokines and measures of disease activity, defined axes/indicators that identified homogeneous patient subgroups by cluster analysis. The new patient classifications resolved major differences between patient subpopulations better than International League of Associations for Rheumatology subtypes. Fourteen variables were identified by sensitivity analysis to crucially determine indicators and clusters. This new schema was conserved in an independent validation cohort. Conclusion Data-driven unsupervised machine learning is a powerful approach for interrogating clinical and biologic data toward disease classification, providing insight into the biology underlying clinical heterogeneity in childhood arthritis. Our analytical framework enabled the recovery of unique patterns from small cohorts and addresses a major challenge, patient numbers, in studying rare diseases.

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

Objective Kawasaki disease (KD) is a multisystem vasculitis affecting children and is characterized by immune activation in the acute stage of disease. Systemic inflammation eventually subsides, although coronary arteritis persists, resulting in aneurysm formation. KD is the leading cause of acquired heart disease among children in North America. Accepted treatment guidelines include high-dose intravenous immunoglobulin (IVIG) and aspirin in the acute phase. Although this therapy is effective, the cellular and molecular mechanisms involved are not clear. The aim of this study was to examine the effect of IVIG and salicylate at each stage of disease development. Methods Using a murine model of KD, we established and validated several in vitro techniques to reflect 3 key steps involved in disease pathogenesis, as follows: thymidine incorporation to evaluate T cell activation, enzyme-linked immunosorbent assay to measure tumor necrosis factor alpha (TNFalpha) production, and real-time polymerase chain reaction to examine TNFalpha-mediated expression of matrix metalloproteinase 9 (MMP-9). Results At therapeutic concentrations, IVIG, but not salicylate, effectively reduced the immune response leading to TNFalpha expression. Unexpectedly, pharmacologic doses of salicylate were not able to inhibit TNFalpha production and in fact enhanced its production. Neither drug directly regulated MMP-9 expression but did so only indirectly via modulating TNFalpha. TNFalpha activity was a prerequisite for local expression of MMP-9 at the coronary artery. Conclusion Therapeutic concentrations of IVIG and salicylate differentially modulate the expression of TNFalpha and its downstream effects. Further dissection of the biologic effects of aspirin in acute KD is necessary for the rational design of therapy.

Objective Kawasaki disease (KD) is a multisystem vasculitis leading to damage in the coronary circulation and aneurysm formation. Because cardiac tissue from affected children is not available, investigation of the mechanisms responsible for coronary artery damage in KD requires use of a disease model. The present study was undertaken to examine, in an experimental model, the role of matrix metalloproteinase 9 (MMP-9) on coronary artery inflammation and vascular damage. Methods C57BL/6 mice were injected with Lactobacillus casei cell wall extract to induce coronary arteritis. Hearts were isolated and assayed for MMP-9 protein expression and enzymatic activity by immunoblotting or confocal microscopy and zymography, respectively. MMP-9–deficient mice were used to examine the necessity of MMP-9 for disease development. Results Localized inflammation at the coronary artery led to elastin breakdown and aneurysm formation. This occurred in the absence of smooth muscle cell apoptosis. Following disease induction, there was an increase in the amount and enzymatic activity of MMP-9, an elastolytic protease. MMP-9 was up-regulated by tumor necrosis factor α (TNFα) and produced primarily by vascular smooth muscle cells. In MMP-9–deficient animals, vascular inflammation continued to develop, but the incidence of elastin breakdown was significantly reduced. Elastin breakdown in the coronary artery was virtually eliminated by ablation of MMP-9. Conclusion These findings show that TNFα up-regulates expression of MMP-9, an important proteinase responsible for extracellular matrix breakdown, leading to coronary artery damage in this model of KD. These results have important implications regarding treatments for improving coronary outcome in affected children.

Kawasaki disease (KD) is a multisystem vasculitis that leads to coronary artery damage and aneurysm formation. Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms. To determine the relationship between circulating levels of elastolytic MMPs and development of coronary artery aneurysms in children with KD, we partnered studies done in affected children with an animal model of disease. In affected children, circulating protein levels and enzymatic activity of MMP-2 and MMP-9 did not have a statistically significant relationship with coronary artery outcome after adjusting for demographic characteristics, and clinical and laboratory features. Although matrix-degrading proteolytic activity was specific for affected mice and localized to inflamed coronary artery segments, the enzymatic activity in the systemic circulation of affected and control mice were not different. Similar to affected children, peripheral blood levels of MMP-9 enzymatic activity did not correlate with coronary artery disease in the animal model. Therefore, circulating levels of MMPs known to act locally may not be useful biomarkers of disease. This is especially relevant to enzymatic activity that is tightly regulated at multiple levels including the local tissue environment.

Kawasaki Disease (KD) is characterized by acute, systemic inflammation followed by a persistent immune response localized to the coronary arteries. Lactobacillus Casei Cell Wall Extract (LCWE) contains among its active ingredients a superantigen (SAg) that induces coronary arteritis in mice. LCWE induces massive immune activation followed by apoptosis or anergy. Previous work has shown that co-stimulatory signals can rescue a subset of SAg-reactive T-cells from apoptosis. CD40-CD40L (ligand) are members of the TNF superfamily which play a role in T-cell co-stimulation. Moreover, genome wide association studies have implicated CD40/CD40L as candidate genes in the pathogenesis of KD. We hypothesize that co-stimulation via the CD40 pathway regulates survival of SAg-reactive T-cells during disease development. Using thymidine proliferation assay and flow cytometry we show that activation of CD40 signaling via agonistic anti-CD40 antibody or recombinant soluble CD40L (rsCD40L) upregulates CD86 (ligand responsible for CD28 signaling) expression, reduces active-caspase-3 expression, indicating decreased cell death and promotes lymphocyte proliferation, Additionally, using CD28 deficient mice we show that lymphocytes do not proliferate in response to SAg plus CD40 stimulation indicating that CD28 is the intermediary that mediates the proliferative effects of CD40 activation. Furthermore, platelets, which are markedly elevated in KD, produce approximately 90% of the soluble CD40L in the circulation. We show that activated platelets (CD62P+) express CD40L and promotes cell survival as evidenced by enhanced lymphocyte proliferation. Likewise, the absence of CD40L negates the proliferative effects of platelet mediated proliferation. Lastly, to determine the role of CD40 stimulation on in vivo development of KD, LCWE was injected into B6 and CD40L deficient mice. We show that inflammation in the coronary arteries does not develop in the absence of CD40L. Therefore, CD40L plays a critical role in immune activation via upregulation of CD86 on antigen presenting cells leading to enhanced co-stimulation and T-cell survival.

The gut microbiome has been shown to have immunomodulatory capabilities, and changes in the composition of the microbiome have altered the pathogenesis of inflammatory diseases. In this study, we sought to investigate the effects of the microbiome on the pathogenesis of Kawasaki Disease (KD) using the Lactobacillus cell wall extract (LCWE)-induced coronary arteritis model. Using qPCR on mouse fecal samples, we quantified bacterial communities through the 16S rRNA gene. Animals originating from the same supplier where housed in two independent facilities. There were marked elevations in Bacteroides, Bifidobacteria, Lactobacillus and segmented filamentous bacteria (SFB) and decrease in Clostridia in Facility A compared to Facility B. The transition to the newer Facility B resulted in a reduction in disease induction by LCWE to 26% (25/95), compared to 50% (35/70) at Facility A. Addition of antibiotics to the drinking water to deplete the microbiome reduced disease incidence to 40% (2/5) of mice housed at Facility A compared to 100% (5/5) of untreated mice, suggesting that components of the microbiome are necessary to exacerbate inflammation. To address whether the gut microbiome was sufficient to support disease susceptibility, the cecal contents of the susceptible mice at A were gavaged into the less susceptible mice at B. Recipients of the cecal contents from Facility A had an increased disease incidence of 91% (10/11) compared to 60% (6/10) in control mice which received cecal contents from mice housed in Facility B. We also report elevated levels of IL-17 in the serum of SFB-colonized mice. SFB, known to exacerbate several models of autoimmunity, was completely absent in mice housed at Facility B. Interestingly Th17 and its associated cytokine IL-17 have been associated with acute KD, but its role in pathogenesis remains unclear. Th17 cells have been implicated in autoimmunity, and are also important players in gut homeostasis. Our data provide evidence for the role of the gut microbiome in modulating the immunopathogenesis of KD, pointing to the important interactions of commensal and pathogenic factors as determinants of disease.

Introduction: Kawasaki Disease (KD) is a multi-system vasculitis leading to coronary artery damage. Previous work has shown that co-stimulatory signals can rescue a subset of superantigen (SAg) reactive T-cells from apoptosis and one of the major pathways responsible for delivery of this co-stimulatory signal is CD28 signaling on T-cells. Lactobacillus casei cell wall extract (LCWE) contains a SAg among its active ingredients, leading to induction of coronary arteritis in mice that closely resembles human KD. Methods: Flow cytometry was used to measure the expression of pro-survival molecules and markers of apoptosis. In vivo studies were performed with C57BL/6 mice (4-5 weeks) injected i.p. with either LCWE, or LCWE and anti-4-1BB or Isotype control antibody. Cardiac tissue isolated, processed, stained and scored as per protocol. Gene expression analysis in KD patients was performed using the Illumina HumanHT-12v4. Results: Despite the fact that SAg-activated T-cells undergo apoptosis and are deleted, T-cells persist and are central to ongoing inflammation in affected arteries. Stimulation of CD28 leads to upregulation of pro-survival molecules cFLIP and BCLxL and reduction of caspase 3-annexinV double positive cells (markers of apoptosis) after SAg-stimulation, as detected by flow cytometry. In animals co-injected with anti-4-1BB (co-stimulation agonist), the incidence of coronary arteritis was dramatically increased to 94% compared to 54% with LCWE alone. Analysis of gene expression profile from 171 children with KD show elevated levels of molecules specific to the CD28 signaling cascade through Grb2, and VAV1 leading to the upregulation of Rac1 and CDC42, together with upregulation of pro-survival molecules cFLIP, MCL1 and NAIP. Interestingly, increased expression of cell survival molecules was associated with IVIG failure, with statistically significant elevation of NAIP and CDC42 and a trend towards increased expression for cFLIP and MCL, in IVIG non-responders compared to responders. Conclusions: Enhanced co-stimulation contributes to T-cell survival after SAg-stimulation leading to persistent coronary artery inflammation and poor treatment response in KD.

Background: ITPKC controls calcium homeostasis and was identified in genome-wide studies to be associated with susceptibility and severity of KD. NLRP3 is activated by danger signals, leading to inflammasome activation and release of IL-1beta and IL-18. Ca 2+ mobilization mediates NLRP3 activation. We studied the contribution of ITPKC and NLRP3 inflammasome activation in KD. Methods: Cytokine levels were measured using ELISA. Gene expression was assayed using Illumina HumanHT-12v4. EBV-transformed B-cell lines from KD patients with different ITPKC genotypes were used to study the gene and protein expression and Ca 2+ flux using qRT-PCR, Western blot analysis, and FACS respectively. In vivo and in vitro analyses from ITPKC-deficient and wildtype mice were performed using ELISA, spinning disc confocal and Westerns, to determine cytokine release, Ca 2+ flux and protein expression responses respectively. Results: Children with KD show increased circulating IL-1beta, IL-18, IL-1RA and IL18BP protein during acute phase of KD (KD n=48, febrile controls n=41 p2+ ] i mobilization resulting in NLRP3 activation and increased production of IL-1beta and IL-18, as demonstrated by gene expression, circulating protein levels and functional assays of PBMCs and immortalized cell lines from affected children. These biologic data are partnered with evidence showing resistance to IVIG therapy in those with the ITPKC polymorphism associated with the highest basal and stimulated [Ca 2+ ] i levels. Studies using ITPKC-deficient mice in an animal model of KD support the molecular, cellular and clinical findings in affected children. Significance: ITPKC regulates NLRP3 activation via control of [Ca 2+ ] i mobilization, directing production of IL-1beta and IL-18, pointing to the key role of calcium mobilization in the immunopathogenesis of KD.

Kawasaki disease is the most common cause of multisystem vasculitis in childhood. The resultant coronary artery lesions make Kawasaki disease the leading cause of acquired heart disease in children in the developed world. TNF-α is a pleiotropic inflammatory cytokine elevated during the acute phase of Kawasaki disease. In this study, we report rapid production of TNF-α in the peripheral immune system after disease induction in a murine model of Kawasaki disease. This immune response becomes site directed, with migration to the coronary arteries dependent on TNF-α-mediated events. Production of TNF-α in the heart is coincident with the presence of inflammatory infiltrate at the coronary arteries, which persists during development of aneurysms. More importantly, inflammation and elastin breakdown in the coronary vessels are completely eliminated in the absence of TNF-α effector functions. Mice treated with the TNF-α-blocking agent etanercept, as well as TNFRI knockout mice, are resistant to development of both coronary arteritis and coronary aneurysm formation. Taken together, TNF-α is necessary for the development of coronary artery lesions in an animal model of Kawasaki disease. These findings have important implications for potential new therapeutic interventions in children with Kawasaki disease.

Kawasaki disease is the most common cause of vasculitis affecting children, and the leading cause of acquired heart disease in the developed world. To date, studies on the role of IFN-γ in the pathogenesis of Kawasaki disease have focused on peripheral production of IFN-γ, and have yielded conflicting results. Affected heart tissue is not available from children with Kawasaki disease. In this study, we use an animal model of Kawasaki disease, Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis, to examine the role of IFN-γ in the development of coronary artery lesions. We report the presence of IFN-γ, both at the mRNA and protein levels, in the affected vessels. Its biphasic expression, first at days 3–7 and again at days 28–42 post-LCWE injection, corresponds to the first appearance of inflammatory infiltrate in coronary arteries, and later to vascular wall disruption and aneurysm formation, respectively. Interestingly, ablation of IFN-γ expression did not dampen the inflammatory response, and IFN-γ-deficient lymphocytes proliferated more vigorously in response to LCWE than those of wild-type animals. Of more importance, the incidence of coronary arteritis was the same in IFN-γ-deficient and wild-type mice. Taken together, our findings demonstrate that IFN-γ regulates the immune response during development of coronary arteritis, but is not required for the induction of coronary artery disease.

Kawasaki disease (KD) is a multisystem vasculitis and the leading cause of acquired heart disease in children of the developed world. Lactobacillus casei cell wall extract-induced coronary arteritis in mice mirrors KD in children. Here, we report that responses to L. casei cell wall extract possess all the hallmarks of a superantigen-mediated response: marked proliferation of naive T cells, non-classical major histocompatibility restriction with a hierarchy in the efficiency of different class II molecules to present this superantigen, a requirement for antigen presentation, but not processing, and stimulation of T cells in a non-clonal, TCR V(beta) chain-dependent fashion. This superantigenic activity directly correlates with the ability to induce coronary arteritis in mice. Taken together, our findings demonstrate that superantigenic activity in L. casei cell wall extract is responsible for induction of coronary artery disease.

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network., Author Summary Differences in gene regulation underlie many important biological processes and are thought to be important for the adaption of organisms to novel environments. Here we focus on the regulation of a group of well-studied genes, the lac operon, that control the utilization of lactose sugar, and we examine how their regulation changes during the adaptation of populations of Escherichia coli bacteria to environments that differ only in the presence of lactose. We find that lac operon regulation is altered in almost all populations that evolve in the presence of lactose and identify two classes of mutations that explain a large part of this change and that confer significant fitness benefits. Interestingly, our study indicates that other mutations, lying outside of the commonly recognized control region, cause new regulation of the lac operon. Together these findings reinforce the importance of changes in gene regulation during evolution and suggest that the biological basis of these changes can be complex and involve novel interactions between genes.

A Superantigen in Lactobacillus casei Cell Wall Extract Induces Coronary Arteritis: An Animal Model for Kawasaki Diseasee

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without beta-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or beta-estradiol (20 ng/ml) or relaxin plus beta-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and beta-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants--a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or beta-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and beta-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and provide evidence for a link between relaxin, MMPs, and matrix degradation.