Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

Mechanisms for Alternaria alternata Function in the Skin During Induction of Peanut Allergy in Neonatal Mice With Skin Barrier Mutations

Neonatal mice with heterozygous mutations in genes encoding the skin barrier proteins filaggrin and mattrin (flaky tail mice [FT+/−]) exhibit oral peanut-induced anaphylaxis after skin sensitization. As we have previously reported, sensitization in this model is achieved via skin co- exposure to the environmental allergen Alternaria alternata (Alt), peanut extract (PNE), and detergent. However, the function of Alt in initiation of peanut allergy in this model is little understood. The purpose of this study was to investigate candidate cytokines induced by Alt in the skin and determine the role of these cytokines in the development of food allergy, namely oncostatin M (Osm), amphiregulin (Areg), and IL-33. RT-qPCR analyses demonstrated that skin of FT+/− neonates expressed Il33 and Osm following Alt or Alt/PNE but not PNE exposure. By contrast, expression of Areg was induced by either Alt, PNE, or Alt/PNE sensitization in FT+/− neonates. In scRNAseq analyses, Osm, Areg, and Il33 were expressed by several cell types, including a keratinocyte cluster that was expanded in the skin of Alt/PNE-exposed FT+/− pups as compared to Alt/PNE-exposed WT pups. Areg and OSM were required for oral PNE-induced anaphylaxis since anaphylaxis was inhibited by administration of neutralizing anti-Areg or anti-OSM antibodies prior to each skin sensitization with Alt/PNE. It was then determined if intradermal injection of recombinant IL33 (rIL33), rAreg, or rOSM in the skin could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE-induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not sufficient for anaphylaxis to oral PNE challenge. Based on these studies a pathway for IL33, Areg and OSM in Alt/PNE sensitized FT+/− skin was defined for IgE induction and anaphylaxis. Alt stimulated two pathways, an IL33 pathway and a pathway involving OSM and Areg. These two pathways acted in concert with PNE to induce food allergy in pups with skin barrier mutations

β-Glucosylceramides and Tocopherols Regulate Development and Function of Dendritic Cells

In humans and mice, offspring of allergic mothers are predisposed to development of allergy. In mice, allergic mothers have elevated β-glucosylceramides (βGlcCers) that are transported to the fetus via the placenta and to offspring via milk. The elevated βGlcCers increase numbers of fetal liver CD11c+CD11b+ dendritic cells (DCs) and offspring allergen-induced lung eosinophilia. These effects are modifiable by maternal dietary supplementation with the plant-derived lipids α-tocopherol and γ-tocopherol. It is not known whether βGlcCers and tocopherols directly regulate development of DCs. In this study, we demonstrated that βGlcCers increased development of GM-CSF-stimulated mouse bone marrow-derived DCs (BMDCs) in vitro without altering expression of costimulatory molecules. This increase in BMDC numbers was blocked by α-tocopherol and potentiated by γ-tocopherol. Furthermore, βGlcCers increased PKCα and PKCδ activation in BMDCs that was blocked by α-tocopherol. In contrast, γ-tocopherol increased BMDC PKCα and PKCδ activation and enhanced the βGlcCer-induced increase in PKCδ activation in a DC subset. Antigen processing per DC was minimally enhanced in βGlcCer-treated BMDCs and not altered ex vivo in lung DCs from pups of allergic mothers. Pups of allergic mothers had an increased proportion of CD11b+CD11c+ subsets of DCs, contributing to enhanced stimulation of T cell proliferation ex vivo. Thus, βGlcCer, which is both necessary and sufficient for development of allergic predisposition in offspring of allergic mothers, directly increased development and PKC activation in BMDCs. Furthermore, this was modifiable by dietary tocopherols. This may inform design of future studies for the prevention or intervention in asthma and allergic disease

Mechanisms for Alternaria alternata Function in the Skin During Induction of Peanut Allergy in Neonatal Mice With Skin Barrier Mutations

Neonatal mice with heterozygous mutations in genes encoding the skin barrier proteins filaggrin and mattrin (flaky tail mice [FT+/−]) exhibit oral peanut-induced anaphylaxis after skin sensitization. As we have previously reported, sensitization in this model is achieved via skin co- exposure to the environmental allergen Alternaria alternata (Alt), peanut extract (PNE), and detergent. However, the function of Alt in initiation of peanut allergy in this model is little understood. The purpose of this study was to investigate candidate cytokines induced by Alt in the skin and determine the role of these cytokines in the development of food allergy, namely oncostatin M (Osm), amphiregulin (Areg), and IL-33. RT-qPCR analyses demonstrated that skin of FT+/− neonates expressed Il33 and Osm following Alt or Alt/PNE but not PNE exposure. By contrast, expression of Areg was induced by either Alt, PNE, or Alt/PNE sensitization in FT+/− neonates. In scRNAseq analyses, Osm, Areg, and Il33 were expressed by several cell types, including a keratinocyte cluster that was expanded in the skin of Alt/PNE-exposed FT+/− pups as compared to Alt/PNE-exposed WT pups. Areg and OSM were required for oral PNE-induced anaphylaxis since anaphylaxis was inhibited by administration of neutralizing anti-Areg or anti-OSM antibodies prior to each skin sensitization with Alt/PNE. It was then determined if intradermal injection of recombinant IL33 (rIL33), rAreg, or rOSM in the skin could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE-induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not sufficient for anaphylaxis to oral PNE challenge. Based on these studies a pathway for IL33, Areg and OSM in Alt/PNE sensitized FT+/− skin was defined for IgE induction and anaphylaxis. Alt stimulated two pathways, an IL33 pathway and a pathway involving OSM and Areg. These two pathways acted in concert with PNE to induce food allergy in pups with skin barrier mutations

Fear of Falling among Older Patients Admitted to Hospital after Falls in Vietnam: Prevalence, Associated Factors and Correlation with Impaired Health-Related Quality of Life

Fear of falling (FOF) diminishes older people’s independence in daily activities, as well as causes serious health and economic consequences. This study examined the prevalence of FOF in older patients hospitalized due to fall-injuries, its effect on health-related quality of life (HRQOL), and its associated factors. We conducted a cross-sectional study in seven hospitals in Thai Binh, Vietnam. FOF was assessed using a single close-ended question. HRQOL was evaluated by the EQ-5D-5L instrument. Multilevel logistic regression and Tobit regression models were utilized. The prevalence of FOF in 405 older patients admitted to hospitals after fall injuries was 88.2%, with a mean EQ-5D index and EQ-VAS of 0.34 (SD = 0.38) and 61.6 (SD = 15.2), respectively. Factors associated with FOF included living alone (OR = 0.13, 95%CI = 0.04; 0.50.,), history of eye diseases (OR = 4.12; 95%CI = 1.91; 8.89), and experiencing psychological distress (OR= 3.56, 95% CI = 1.05; 12.00). After adjusting for confounders, the EQ-5D index in the FOF group reduced by 0.15 points (Coef. = −0.15; 95%CI= −0.24; −0.05) compared to that of non-FOF group. Our study shows that FOF had an independent negative relationship with HRQOL of patients. Improving knowledge about fall prevention in patients and caregivers could reduce the burden of falls in older people

Health-Related Quality of Life Profiles among Patients with Different Road Traffic Injuries in an Urban Setting of Vietnam

Road traffic injuries (RTIs) cause a substantial disease burden in Vietnam. Evaluating health-related quality of life (HRQOL) among patients having a diversity of RTIs informs an integral part of treatment effectiveness. This study aims to examine HRQOL of patients suffering different RTIs in Vietnam’s urban areas. A cross-sectional study was conducted on 408 patients from October to December 2018 in six hospitals in Thai Binh. The EuroQol-5 dimensions-5 levels (EQ-5D-5L) and visual analog scale (VAS) were used to assess the HRQOL of patients. Multivariable Tobit regression was applied to measure the difference of HRQOL among different kinds of injuries. The mean EQ-5D-5L and VAS score was 0.40–0.66, respectively. Mean EQ-5D-5L index was lowest in patients with oral and facial injuries (0.22) and fracture injuries (0.23), while patients having hand injuries had the highest EQ-5D-5L index (0.54). EQ-5D-5L index had a negative association with oral, facial, and fracture injuries. Meanwhile, patients with brain, fracture, and multiple injuries tended to have lower VAS score. Poor HRQOL among patients injured in road traffic were observed. Pain management, early rehabilitation, and mental health counseling services should be considered during treatment time, especially among those having the brain, oral and facial trauma, fracture, and multiple injuries

Impact of a Smoking Cessation Quitline in Vietnam: Evidence Base and Future Directions

Smoking is considered the most critical modifiable factor with regard to lung cancer and remains a public health concern in many countries, including Vietnam, which is among those countries with the highest tobacco consumption rates in the world. This study has examined the impact of national telephone counselling for smoking cessation and has identified the factors associated with the impact of the quitline among male callers in Vietnam. A randomized cross-sectional survey of 469 smokers who sought smoking cessation services via the national quitline was performed from September 2015 to May 2016. The primary outcomes were measured by a self-reported quit rate at the time of assessment, 7 day point prevalence abstinence (PA), 6 month prolonged PA, service satisfaction, and level of motivation. Among the participants, 31.6% were abstinent, and 5.1% of participants successfully stopped smoking and did not need to seek quitline support. Most of the clients were satisfied with the quality of service (88.5%), felt more confident about quitting (74.3%), and took early action via their first quit attempt (81.7%); 18.3% reported a more than 7 day abstinence period at the time of survey. The primary reasons for smoking relapse were surrounding smoking environments (51.6%) and craving symptoms (44.1%). Future smoking cessation efforts should focus on improving the quality of quitline services, client satisfaction, and developing a tailored program and counseling targeting smokers with specific characteristics, especially ones experiencing chronic diseases