Diagnostic value of DNA or RNA-based metagenomic next-generation sequencing in lower respiratory tract infections.

Objectives: We aimed to evaluate and compare the diagnostic performance of RNA-mNGS and DNA-mNGS workflow in bacterial pneumonia, fungal pneumonia and tuberculosis. Methods: A total of 134 cases suspected pneumonia undergoing both DNA and RNA based mNGS of bronchoalveolar lavage fluid (BALF) and also traditional etiological examination were evaluated retrospectively.Sensitivity, specificity, PPV, NPV and accuracy rate of DNA and RNA based mNGS were estimated. Results: In the diagnosis performance of bacterial pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS(82.3 % vs. 61.9 %, P < 0.01). There was no significant difference of sensitivity between the two process(71.4 % vs. 85.7 %, P = 0.375).In the diagnosis performance of fungal pathogens in LRTIs,the specificity of RNA-mNGS was higher than that of DNA-mNGS (72.3 % vs. 27.3 %,p < 0.001). There was no significant difference of sensitivity between the two process(96.5 % vs. 98.8 %,p = 0.125).In the diagnosis performance of tuberculosis in LRTIs,the sensitivity of DNA-mNGS was higher than that of RNA-mNGS (91.7 % vs. 33.3 %,p = 0.016),the specificity was similar in the two process (100 %). Conclusions: RNA-mNGS may reduced the misdiagnosis rate of bacterial and fungal pathogens in LRTIs.Compared to RNA-mNGS, DNA-mNGS may could improve the diagnostic rate of tuberculosis.

Distinct roles of PI3Kδ and PI3Kγ in a toluene diisocyanate-induced murine asthma model.

TDI-induced asthma is characterized by neutrophil-dominated airway inflammation and often associated with poor responsiveness to steroid treatment. Both PI3Kδ and PI3Kγ have been demonstrated to play important proinflammatory roles in ovalbumin-induced asthma. We've already reported that blocking pan PI3K effectively attenuated TDI-induced allergic airway inflammation. Yet the specific functions of PI3Kδ and PI3Kγ in TDI-induced asthma are still unclear. Male BALB/c mice were first dermally sensitized and then challenged with TDI to generate an asthma model. Sellective inhibitors of PI3Kδ (IC-87114, AMG319) and PI3Kγ (AS252424, AS605240) were respectively given to the mice after each airway challenge. Treatment with IC-87114 or AMG319 after TDI exposure led to significantly decreased airway hyperresponsiveness (AHR), less neutrophil and eosinophil accumulation, attenuated airway smooth muscle (ASM) thickening, less M1 and M2 macrophages in lung, as well as lower levels of IL-4, IL-5, IL-6 and IL-18 in bronchoalveolar lavage fluid (BALF) and recovered IL-10 production. While mice treated with AS252424 or AS605240 had increased AHR, more severe ASM thickening, larger numbers of neutrophils and eosinophils, more M1 but less M2 macrophages, and higher BALF levels of IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 when compared with those treated with vehicle. These data revealed that pharmacological inhibition of PI3Kδ attenuates TDI-induced airway inflammation while PI3Kγ inhibition exacerbates TDI-induced asthma, indicating distinct biological functions of PI3Kδ and PI3Kγ in TDI-induced asthma.

Protective effects of N-acetylcysteine on a chemical-induced murine model of asthma.

INTRODUCTION: Oxidative stress is involved in the pathophysiology of inflammatory airway diseases, including asthma. In this study, we elucidated the possible protective effects of the antioxidant N-acetylcysteine (NAC) on a toluene diisocyanate (TDI)-induced murine asthma model. METHODS: Male BALB/c mice were sensitized and challenged with TDI to generate a chemical-induced asthma model. NAC was given intraperitoneally to mice immediately after each TDI challenge. Airway reactivity to methacholine and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology. RESULTS: NAC treatment dramatically reduced the increased airway hyperresponsiveness, inflammatory infiltration, and goblet cell metaplasia in TDI-exposed mice. Numbers of total cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of TDI-challenged mice were significantly higher than vehicle control, but the administration of NAC decreased these inflammatory cell counts. TDI exposure led to significantly increased levels of interleukin 4 (IL-4) and IL-5, which were also suppressed by NAC. In addition, diminished lung reduced oxidized glutathione ratio and superoxide dismutase activity were observed after TDI challenge, and these changes were attenuated by NAC. CONCLUSION: NAC treatment has beneficial effects in TDI-induced asthma.

[Analysis of the positive rate of anal swab nucleic acid test and clinical characteristics in patients with different severity of coronavirus disease 2019].

OBJECTIVE: To compared the positive rate of anal swab nucleic acid test and clinical characteristics of critical and general coronavirus disease 2019 (COVID-19) patients. METHODS: Clinical data of 18 patients with COVID-19 admitted to the First People's Hospital of Lianyungang City from February to March 2020 were retrospectively analyzed. The patients were divided into general group (n = 11) and critical ill group (n = 7) according to the severity of the disease. The differences of gender, age, epidemiological characteristics, fever duration after admission, underlaying disease, positive rate of anal swab nucleic acid test at admission and two times of negative pharyngeal swab test were compared between the two groups. RESULTS: There were no significant differences in gender, age, fever duration after admission or underlaying disease between the two groups. The number of anorectal swab positive cases in critically ill group was significantly higher than that in general group (cases: 4 vs. 1, P = 0.047). After two negative pharyngeal swab nucleic acid test, the number of anal swab positive cases in critical illness group was still higher than that in general group (cases: 2 vs. 0), but the difference was not statistically significant (P = 0.137). The number of non-local infection in critical ill group was significantly higher than that in general group (cases: 4 vs. 0, P = 0.047). All of the 4 non-local infected patients had a history of living in Wuhan. CONCLUSIONS: The patients with anorectal swab nucleic acid positive may have a more serious condition. It may be a risk to transfer ill patients out of the isolation ward by the criteria of only two times of negative pharyngeal swab nucleic acid test. Patients returning to our city after infection in Wuhan may be more serious.

Survival after cardiac arrest secondary to high-risk pulmonary embolism without reperfusion therapies: A case report.

INTRODUCTION: High-risk pulmonary embolism (PE) needs reperfusion therapies. However, it is difficult to make medical decisions when thrombolysis is contraindicated, though pulmonary embolectomy and percutaneous catheter-directed treatment (CTD) are recommended for these patients. PATIENT CONCERNS: We reported here a case of high-risk PE patient with cardiac arrest (CA), vertebral compression fracture, as well as scalp and frontal hematoma. DIAGNOSIS: The diagnosis of PE was based on computed tomography pulmonary angiography (CTPA) which demonstrated filling defects in the right and left pulmonary arteries. INTERVENTIONS: Cardiopulmonary resuscitation was performed until the patient returned to idioventricular rhythm 3 minutes after admitted. She suffered another half-hour of hemodynamic disturbance after her shock improved 3 days later. The diagnosis of PE was confirmed by CTPA at that time. The patient did not receive any reperfusion therapies because hemoglobin decreased significantly. Moreover, anticoagulation was postponed for 2 weeks when bleeding appeared to be stopped. She received overlapping treatment with low molecular weight heparin and warfarin for 5 days then warfarin alone and discharged. OUTCOMES: She was discharged with normal vital signs and neurologically intact. She received anticoagulant therapy with warfarin and international normalized ratio regularly monitored after she was discharged, moreover, the pulmonary artery pressure turned normal, as determined by transthoracic echocardiography 1 month later. The warfarin treatment was discontinued after 12 months and no evidence of recurrence was seen until recently. CONCLUSIONS: This is the first case report of PE combined with CA that did not receive reperfusion therapy. We hypothesized that there was a spontaneous resolution in pulmonary emboli.

Chicken IgY facilitates allergic airway inflammation in a chemical-induced murine asthma model by potentiating IL-4 release.

High mobility group box 1 (HMGB1) is a DNA-binding protein that is abundantly expressed in most tissues. Recently, HMGB1 has gained much attention for its regulation of immunity and inflammation. Yet its role in toluene diisocyanate (TDI)-induced asthma still remains poorly characterized. In this study, mice were sensitized and challenged with TDI to establish a TDI-induced asthma model. An IgY anti-HMGB1 antibody or isotype IgY was given intraperitoneally after each challenge. Airway reactivity to methacholine, airway inflammation, bronchial epithelial hyperplasia and shedding were unexpectedly aggravated after administration of the anti-HMGB1 antibody and was accompanied by increased pulmonary expression of HMGB1, especially in those mice treated with IgY. Levels of IL-4, IL-5, IL-13 and TNF-α were also elevated with TDI-induction. Primary lymphocytes from TDI sensitized and challenged mice demonstrated increased secretion of IL-4 after IgY stimulation. To confirm the effect of IgY, a cohort of mice exposed to TDI or vehicle was injected with IgY and the same results were observed after IgY treatment as in TDI asthmatic mice. Taken together, these results show that the IgY anti-HMGB1 antibody can facilitate TDI-induced allergic airway inflammation. Specifically, IgY, rather than anti-HMGB1, plays an important role in the process of exacerbated asthma, shedding light on an underappreciated role of avian IgY.

Phosphatidylinositol 3-Kinase Mediates ß-Catenin Dysfunction of Airway Epithelium in a Toluene Diisocyanate-Induced Murine Asthma Model.

Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Yet the role of phosphatidylinositol 3-kinase (PI3K) in dysregulation of airway epithelial adherens junctions in toluene diisocyanate (TDI)-induced asthma has not been addressed. Male BALB/c mice were first dermally sensitized and then challenged with TDI by means of compressed air nebulization. The mice were treated intratracheally with PI3K inhibitor LY294002. Levels of phospho-Akt in airway epithelium and whole lung tissues were markedly increased in TDI group compared with control mice, which decreased after administration of LY294002. The dilated intercellular spaces of airway epithelium induced by TDI were partially recovered by LY294002. Both the protein expression and distribution of adherens junction proteins E-cadherin and ß-catenin were altered by TDI. Treatment with LY294002 rescued the distribution of E-cadherin and ß-catenin at cell-cell membranes, restored total ß-catenin pool, but had no effect on protein level of E-cadherin. At the same time, LY294002 also inhibited phosphorylation of ERK, glycogen synthase kinase3ß and tyrosine 654 of ß-catenin induced by TDI. In summary, our results showed that the PI3K pathway mediates ß-catenin dysregulation in a TDI-induced murine asthma model, which may be associated with increased tyrosine phosphorylation of ß-catenin.

1,25-Dihydroxyvitamin D3 prevents toluene diisocyanate-induced airway epithelial barrier disruption.

The loss of airway epithelial integrity contributes significantly to asthma pathogenesis. Evidence suggests that vitamin D plays an important role in the prevention and treatment of asthma. However, its role in airway epithelial barrier function remains uncertain. We have previously demonstrated impaired epithelial junctions in a model of toluene diisocyanate (TDI)-induced asthma. In the present study, we hypothesized that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] may prevent TDI-induced epithelial barrier disruption. Male BALB/c mice were dermally sensitized and then challenged with TDI. The mice were then administered 1,25(OH)2D3 intraperitoneally prior to challenge with TDI. For in vitro experiments, 16HBE bronchial epithelial cells were cultured and stimulated with TDI-human serum albumin (HSA). The results revealed that the mice treated with 1,25(OH)2D3 displayed decreased airway hyperresponsiveness (AHR), suppressed neutrophil and eosinophil infiltration into the airways, as well as an increased E-cadherin and zonula occludens-1 (ZO-1) expression at the cell-cell contact sites. In vitro, exposure of the cells to TDI-HSA induced a rapid decline in transepithelial electrical resistance (TER) and an increase in cell permeability, followed by a decrease in occludin expression and the redistribution of E-cadherin, accompanied by a significant upregulation in the levels of phosphorylated extracellular signal-regulated kinase (ERK)1/2. These effects were all partly reversed by treatment with either 1,25(OH)2D3 or an ERK1/2 inhibitor. In conclusion, the findings of our study demonstrate that 1,25(OH)2D3 prevents TDI-induced epithelial barrier disruption, and that the ERK1/2 pathway may play a role in this process.

Ethyl pyruvate decreases airway neutrophil infiltration partly through a high mobility group box 1-dependent mechanism in a chemical-induced murine asthma model.

BACKGROUND: Diisocyanates are one of the leading causes of occupational asthma, which is dominated by granulocytic inflammation in the airway. In this study, we intended to explore the role of ethyl pyruvate (EP) on neutrophil infiltration in a toluene-2,4-diisocyanate (TDI)-induced murine asthma model. METHODS: The experimental mice were first dermally sensitized and then challenged with TDI via oropharyngeal aspiration. The mice were treated intraperitoneally with 100, 50 or 10mg/kg EP 1h before each challenge. One day after the last challenge, airway reactivity to methacholine was measured by a barometric plethysmographic chamber. Total and differential cell counts, along with levels of macrophage inflammatory protein-2 (MIP-2), TNF-α in bronchoalveolar lavage (BAL) fluid and mRNA expression of CXCR2 in the lung were assessed. To depict neutrophils, a naphthol AS-D chloroacetate esterase kit was used. High mobility group box 1 (HMGB1) was determined by western blot and immunohistochemistry. RESULTS: Treatment with EP dramatically decreased airway hyperresponsiveness in TDI-challenged mice, as well as numbers of neutrophils in BAL fluid and peribronchovascular regions. Both the TDI-induced raised protein level and abnormal distribution of HMGB1 were significantly recovered by EP in a dose-dependent manner. The concentration of MIP-2 in TDI-induced asthma mice was significantly higher than that of the control ones, while EP had few effects on MIP-2. The mRNA expression of CXCR2 didn't change significantly, and TNF-α was not detected in BAL fluids. CONCLUSION: EP reduces airway neutrophil infiltration partly through downregulating HMGB1 in a chemical-induced murine asthma model.

Induction of thymic stromal lymphopoietin expression in 16-HBE human bronchial epithelial cells by 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3.

Vitamin D exerts profound effects on airway epithelial cells. Thymic stromal lymphopoietin (TSLP) derived from airway epithelial cells plays a role in the innate and antigen­specific adaptive immune responses. However, the effect of vitamin D on TSLP expression in airway epithelial cells is unclear. In this study, 16-HBE human bronchial epithelial (HBE) cells were cultured with various concentrations of 25-hydroxyvitamin D(3) (25 D(3)) and 1,25-dihydroxyvitamin D(3) (1,25 D(3)). The expression of TSLP in the 16-HBE human bronchial epithelial cell line was analyzed by PCR and enzyme-linked immunosorbent assay (ELISA). We found that the 16-HBE cells converted inactive 25 D(3) to active 1,25 D(3) and that TSLP mRNA and protein expression levels were significantly increased, peaking at 2 or 12 h in the cells exposed to 500 nM 25 D(3) and 50 nM 1,25 D(3) respectively. Since vitamin D(3) upregulated protein 1 (VDUP1) plays a multifunctional role in a variety of cellular responses, we hypothesized that VDUP1 is involved in the induction of TSLP production by 25 D(3). The results showed that the mRNA and protein levels of VDUP1 were significantly upregulated by vitamin D. Furthermore, the silencing of VDUP1 by small interfering RNA (siRNA) significantly inhibited the 25 D(3)- and 1,25 D(3)-mediated induction of TSLP expression. To characterize the metabolic properties of vitamin D in airway epithelial biology, we used the chemical inhibitor of 1α-hydroxylase, itraconazole. The results revealed that itraconazole (10-6 M) reduced the 25 D(3)- but not the 1,25 D(3)-induced TSLP expression in 16-HBE cells. Based on these data, it can be concluded that vitamin D increases TSLP expression in 16-HBE cells through the VDUP1 pathway, which suggests a novel mechanism by which vitamin D alters immune function in the lungs.

Mechanism of E-cadherin redistribution in bronchial airway epithelial cells in a TDI-induced asthma model.

E-cadherin (epithelial cadherin), a transmembrane protein, provides essential architecture and immunological function to the airway epithelium, a barrier structure that plays an essential role in asthma pathogenesis. Toluene diisocyanate (TDI) is currently one of the leading causes of occupational asthma. However, relatively few studies have been undertaken to determine the biological effects of TDI on the barrier properties of airway epithelium, but it is known that TDI can damage airway epithelial tight junctions in vitro. Here, we hypothesize that TDI can injure E-cadherin both in normal and allergic-induced airway epithelium. To test this, we developed a murine model of TDI-induced asthma characterized by neutrophil-dominated airway inflammation, epithelial shedding, and obvious aberrant distribution of E-cadherin. Pretreatment with dexamethasone (DEX) significantly rescued the immunoreactivity of E-cadherin, accompanied by increased neutrophils in bronchoalveolar lavage fluid (BALF). In vitro, TDI-human serum albumin (HSA)-induced redistribution of E-cadherin was associated with extracellular signal-regulated kinase (ERK)1/2 activation. The inhibition of phospho-ERK (p-ERK)1/2 by DEX can partly reverse this reaction. These results indicate that E-cadherin redistribution may be an important contributor in the generation of TDI-induced asthma.

[High-mobility group box protein 1 in synergy with interleukin-1ß promotes interleukin-8 expression in human airway epithelial cells in vitro].

OBJECTIVE: To test the effect of high-mobility group box protein 1 (HMGB1) alone or in synergy with interleukin-1ß (IL-1ß) on the expression of IL-8 in human airway epithelial cells in vitro. METHODS: Human airway epithelial 16HBE and A549 cell lines were incubated with HMGB1 (100 ng/ml) in the absence or presence of IL-1ß (10 ng/ml) for 24 h, and the changes of IL-8 mRNA and protein expressions were assessed using quantitative PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: In the two human airway epithelial cell lines, HMGB1 alone did not produce obvious effect on the expression of IL-8, but in the presence of IL-1ß, HMGB1 caused a significant increase of IL-8 expressions at both the mRNA and protein levels. CONCLUSION: HMGB1 in synergy with IL-1ß increases the expression of IL-8 in human airway epithelial cells, which provides new evidence that HMGB1 contributes to neutrophilic airway inflammation by regulating IL-8 expression.