Bronchial Alveolar Lavage Fluid Research Articles

Ferruginous components of particulate matters in subway environments, α-Fe2O3 or Fe3O4, exacerbates allergies

The respiratory effects of particulate matter (PM) in subway station platforms or tunnels have attracted considerable research attention. However, no studies have characterized the effects of subway PM on allergic immune responses. In this study, iron oxide (α-Fe2O3 and Fe3O4) particles—the main components of subway PM—were intratracheally administered to BALB/c mice where ovalbumin (OVA) induced allergic pulmonary inflammation. Iron oxide particles enhanced OVA-induced eosinophil recruitment around the bronchi and mucus production from airway epithelium. The concentrations of type 2 cytokines, namely, interleukin (IL)-5 and IL-13, in bronchial alveolar lavage fluids were increased by iron oxide particles. Iron oxide particles also increased the number of type 2 innate lymphoid cells and CD86+ cells in the lung. Moreover, phagocytosis of particles in lung cells was confirmed by Raman spectroscopy. In a subsequent in vitro study, bone marrow-derived antigen-presenting cells (APCs) isolated from NC/Nga mice were exposed to iron oxide particles and OVA. They were also exposed to outdoor ambient PM: Vehicle Exhaust Particulates (VEP) and Urban Aerosols (UA) as references. Iron oxide particles promoted the release of lactate dehydrogenase, C-X-C motif chemokine ligand 1 and IL-1α from APCs, which tended to be stronger than those of VEP. These results suggest that iron oxide particles enhance antigen presentation in the lungs, promoting allergic immune response in mice; iron oxide particles-induced death and inflammatory response of APCs can contribute to allergy exacerbation. Although iron oxide particles do not contain various compounds like VEP, iron oxide alone may have sufficient influence.

Allergic bronchopulmonary mycosis due to Schizophyllum commune in a patient with chronic hepatitis B.

Schizophyllum commune (S. commune) is an opportunistic pathogenic fungus and can cause infection of the respiratory system in immunocompromised hosts. Allergic bronchopulmonary mycosis (ABPM) is the major disease caused by S. commune. However, identification of S. commune using routine mycological diagnostic methods is difficult. It is easy to make mistakes in diagnosis and treatment, resulting in deterioration of the disease. We report the first case of ABPM due to S. commune in a Chinese patient with chronic hepatitis B. The patient presented cough, sputum and dyspnea for six months. The pathogen was missed during routine laboratory workup. We performed bronchoscopy examination and bronchoalveolar lavage. S. commune was identified by metagenomic next-generation sequencing (mNGS) of bronchial alveolar lavage fluid (BALF). Hence, the patient was immediately treated with 200 mg voriconazole twice daily (intravenous infusion) and 20 mg prednisone once a day (oral therapy), along with oral entecavir for hepatitis B. There was no recurrence of infection after the medication was discontinued. S. commune infection should be considered in the diagnosis of patients with refractory cough, sputum and dyspnea, especially in immunocompromised individuals. The mNGS technique is an effective supplementary technique for the diagnosis of S. commune infection, enabling precise clinical decision-making and appropriate treatment. Most patients have good prognosis with a combination of proper antifungal therapy and hormonal therapy.

6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway

BackgroundAlveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. MethodsRat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). Results6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKβ/total IKKβ, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. ConclusionsOur experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.

Kechuanning Gel Plaster Exerts Anti-inflammatory and Immunomodulatory Effects on Ovalbumin-induced Asthma Model Rats via ERK Pathway.

We aimed to evaluate the therapeutic effects of Kechuanning gel plaster on ovalbumin (OVA)-induced rat model of asthma. Rats were injected with OVA to induce asthma, and Kechuanning gel plaster was administered after the OVA challenge. The immune cell counts in the bronchial alveolar lavage fluid (BALF) were calculated after Kechuanning gel plaster administration. The levels of immune factors in BALF and serum OVA-specific IgE levels were analyzed. Western blot analysis and immunohistochemistry were carried out to analyze the following proteins: C-FOS, C-JUN, RAS p21 protein activator 1 (RASA1), matrix metalloproteinase 9 (MMP9), RAF1, p-MEK1, tissue inhibitor of metalloproteinase-1 (TIMP1), and p-extracellular signal-regulated kinase 1 (ERK1). Administration of Kechuanning gel plaster led to decreased immune cell counts, inflammatory cytokines (interleukin (IL)-1β, IL13, and IL17), and OVA-specific IgE expression. Compared to the normal group, the C-FOS, C-JUN, RASA1, MMP9, RAF1, MEK1, TIMP1, and p- ERK1 expressions in the model group were significantly increased, whereas Kechuanning gel plaster administration decreased C-JUN, MMP9, TIMP1, RAF1, MEK1, p-ERK1, C-FOS, and RASA1 protein levels. Kechuanning gel plaster exerted its therapeutic effects on OVA-induced asthma model rats through the ERK signaling pathway. Kechuanning gel plaster could be considered as a potential alternative therapeutic agent for the management of asthma.

Investigation of SARS-CoV-2 individual proteins reveals the in vitro and in vivo immunogenicity of membrane protein

Evidence in SARS-CoV-2 patients have identified that viral infection is accompanied by the expression of inflammatory mediators by both immune and stromal cells within the pulmonary system. However, the immunogenicity of individual SARS-CoV-2 proteins has yet to be evaluated. The SARS-CoV-2 virus consists of 29 proteins, categorized either as nonstructural proteins (NSP’s), structural proteins (SP’s) or accessory proteins. Here we sought to evaluate the immunogenicity of NSP 1, 7, 8, 9, 10, 12, 14, 16 and the SP’s spike protein (full length, S1, S2 and receptor binding domain subunits), nucleocapsid and membrane SARS-CoV-2 proteins against THP-1 and human peripheral blood mononuclear cells (PBMCs). Our results indicate that various SARS-CoV-2 proteins elicit a proinflammatory immune response indicated by increases in cytokines TNF, IL-6 and IL-1β. Our results support that SARS-CoV-2 membrane protein induced a robust increase of TNF, IL-6, IL-1β and IL-10 expression in both THP-1 and human PBMC’s. Further evaluation of intranasal membrane protein challenge in male and female BALB/c mice show increases in BALF (bronchalveolar lavage fluid) proinflammatory cytokine expression, elevated cellularity, predominantly neutrophilic, and concomitant peribronchiolar and perivascular lymphomononuclear and neutrophilic inflammation. Our results suggest that individual membrane associated SARS-CoV-2 proteins induce a robust immune response that may contribute to viral induced cytokine release syndrome (CRS) in the lungs of moderate to severe COVID-19 patients. We posit that SARS-CoV-2 membrane challenges in immune-competent mice can serve as an adequate surrogate for the development of novel treatments for SARS-CoV-2 induced pulmonary inflammation, thereby avoiding expensive live virus studies under BSL-3 conditions.

Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2).MethodsIn this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis.ResultsElevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-β upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion.ConclusionsThese findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Application Value of Next-Generation Sequencing of Bronchial Alveolar Lavage Fluid in Emergency Patients with Infection.

This study was performed to explore the application value of next-generation sequencing (NGS) of bronchial alveolar lavage fluid (BALF) in emergency patients with infection. In this regard, a total of 52 patients with infection who were diagnosed and treated in the emergency department of our hospital from September 2019 to September 2021 were selected as the research objects. The BALF of the patients was analyzed by NGS, and the results were compared with the pathogen detection results of traditional microbial culture of the patients to analyze the diagnostic value of NGS in patients with infection. The results showed that among the 52 patients, 47 were positive by NGS and 13 were positive by traditional microbial culture. The pathogen detection rate of NGS was higher than that of traditional microbial culture[90.4% (47/52) VS 25% (13/52), χ2=45.539, P<0.001], and was able to detect viruses, fungi and other special pathogens that were difficult to be detected by traditional microbial culture, such as Chlamydia psittaci. A total of 129 pathogens were detected in the NGS test results of 47 of these patients, including bacteria, fungi, viruses and mycoplasma/chlamydia. 14 pathogens were detected in the conventional microbiological cultures of 13 patients, including bacteria and fungi. Overall, compared with traditional microbial culture methods, NGS detection has higher accuracy and can effectively detect pathogens that cannot be detected by traditional microbial culture. It has a high application value in the diagnosis of pathogens and can provide clinical guidance for the diagnosis of pathogens in patients with emergency infections.

Human placenta-derived mesenchymal stem cell administration protects against acute lung injury in a mouse model.

This study aims to investigate the effect of placenta-derived mesenchymal stem cells (PMSCs) administration on tissue repair following acute lung injury (ALI). PMSCs were transplanted intravenously to a mouse model of lipopolysaccharide-induced ALI. The therapeutic effects were determined by evaluating several indicators, including pathology; the wet/dry ratio of the lungs; blood gas analysis; the total protein content, cell numbers, and the activity of myeloperoxidase (MPO) in bronchial alveolar lavage fluid (BALF); and the levels of anti-inflammatory and proinflammatory cytokines in serum and BALF. To investigate the underlying mechanism, PMSC-derived exosomes were used for ALI treatment. Administration of PMSCs improved the degree of lung injury, reduced inflammation, increased the expression levels of anti-inflammatory cytokines, and protected lung function. As expected, the effects of PMSC-derived exosomes in the ALI model were similar to those of PMSCs, both in terms of improved lung function and reduced inflammation. These findings suggest that PMSCs have ameliorating effects on ALI that are potentially mediated via their secreted exosomes.

ADP Ribosylation Factor 6 Relieves Airway Inflammation and Remodeling by Inhibiting Ovalbumin Induced-Epithelial Mesenchymal Transition in Experimental Asthma, Possibly by Regulating of E2F Transcription Factor 8

ABSTRACT Background Childhood asthma is a major global health concern. ADP-ribosylation factor 6 (ARF6) is a low-molecular-weight GTPase; however, its role in childhood asthma remains unclear. Methods Ovalbumin (OVA)-challenged neonatal mice and transforming growth factor-β1 (TGF-β1)-induced BEAS-2B cells were used as in vivo and in vitro models of childhood asthma, respectively. Results Upon OVA stimulation, ARF6 expression was upregulated in the lung tissue. Neonatal mice administered SehinH3 (an ARF6 inhibitor) exhibited improved pulmonary pathological injury, along with reduced inflammatory cell infiltration in the lungs and cytokine release in bronchial alveolar lavage fluid and serum (interleukin [IL]−3, IL−5, IL−13, IgE, and OVA-specific IgE). SehinH3 treatment restrained epithelial – mesenchymal transition (EMT) in the lungs of asthmatic mice, as evidenced by increased E-cadherin and decreased N-cadherin and α-smooth muscle actin expression. Different TGF-β1 exposures to BEAS-2B cells induced a time- and dose-dependent increase in ARF6 expression in vitro. Upon TGF-β1 stimulation, ARF6 knockdown repressed EMT and SehinH3 treatment caused similar results in BEAS-2B cells. The transcription factor E2F8 is involved in diverse biological functions and its increased expression was confirmed in vivo and in vitro. Dual-luciferase assays confirmed that E2F8 binds to the ARF6 promoter and promotes its transcriptional activity. In vitro results revealed that E2F8 silencing suppressed EMT, whereas rescue experiments showed that ARF6 overexpression partly reversed these phenomena. Conclusion Our study showed that ARF6 is associated with childhood asthma progression and may be positively regulated by E2F8. These results provide insight into the pathogenesis and treatment of childhood asthma.

Early lyophilized cryoprecipitate enhances the ADAMTS13/VWF ratio to reduce systemic endotheliopathy and lessen lung injury in a mouse multiple-trauma hemorrhage model.

Recent studies in severely injured patients suggest an important role of von Willebrand Factor (VWF) and ADAMTS13 in the endotheliopathy of trauma (EoT). We hypothesized that the early use of cryoprecipitate would be effective as an endothelial protector by supplementing physiologic VWF and ADAMTS13 to reverse the EoT. We tested a pathogen-reduced lyophilized cryoprecipitate (LPRC) that could expedite the early administration of cryoprecipitate in the battlefield. A mouse multiple-trauma model with uncontrolled hemorrhage (UCH) from liver injury was utilized followed by hypotensive resuscitation (mean arterial pressure, 55-60) × 3 hours with lactated Ringer's (LR), fresh frozen plasma (FFP), conventional pathogen-reduced cryoprecipitate (CC), and LPRC. Blood was collected for measurement of syndecan-1, VWF, and ADAMTS13 by ELISA. Lungs were stained for histopathologic injury and syndecan-1 and bronchial alveolar lavage (BAL) fluid harvested for protein as an indicator of permeability. Statistical analysis was by ANOVA followed by Bonferroni correction. Following multiple trauma and UCH, blood loss was similar across groups. Mean volume of resuscitation was higher in the LR group compared with the other resuscitation groups. Lung histopathologic injury, syndecan-1 immunostaining and BAL protein were higher with LR compared with resuscitation with FFP and CC, while LPRC further reduced BAL compared with FFP and CC. The ADAMTS13/VWF ratio was significantly lower in LR but improved with FFP and CC, comparable to shams while LPRC further increased this ratio. The protective effects of CC and LPRC were comparable to FFP in ameliorating the EoT in our murine multiple trauma and UCH model. Lyophilized cryoprecipitate may also provide additional benefit by enhancing the ADAMTS13/VWF ratio. These data provide evidence of the safety and efficacy of LPRC and warrants further investigation for its potential application in military settings once approved for human administration.

Commensal probiotic provides long-term protection in a murine model of allergic lung disease by altering the host microbiota

Abstract The incidence of allergic diseases is increasing, with house dust mite allergy, afflicting up to 20% of the population in developed countries. House dust mites (HDM) are a common allergen that can be difficult to avoid and triggers allergic reactions, including wheezing and shortness of breath. Bacillus subtilis is a gut-associated commensal probiotic that can prevent murine HDM-induced allergic lung inflammation. We exposed C57Bl/6 mice to B. subtilis via oral gavage and challenged with intranasal HDM for up to 8 weeks. We found that pretreatment with B. subtilis was able to provide protection from eosinophil infiltration, measured in bronchial alveolar lavage fluid (BALF), for 3 weeks. This loss of protection correlated with an increase in the eosinophil chemoattractant CCL24 levels in the BALF and reduced abundance of the bacterial phylum Verrucomicorbiota in collected feces. Additionally, we found that B. subtilis treatment altered the bacterial composition by increasing the abundance of the bacteria phylum Bacteroidetes. These results support B. subtilis as a prophylactic for preventing allergic lung disease and highlights that protection can last up to 3 weeks. This work also expands our understanding of how B. subtilis mediates protection and that in addition to modifying the immune system, it is also altering the host microbiota. This work is supported by start-up funds and core facility outsourcing funds from Midwestern University.

Prediction of target genes in community-acquired pneumonia based on the bioinformatics method.

To screen the related genes of community-acquired pneumonia (CAP) by bioinformatics technology, and to analyze the clinical value of key genes. Gene chip data sets containing CAP patients and normal controls were screened from the Gene Expression Omnibus (GEO) database. The downregulated differentially expressed genes (DEGs) were screened using a gene expression analysis tool (GEO2R). Simultaneously, gene set enrichment analysis (GSEA) was used to explore the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and core genes related to CAP. The candidate genes were then intersected with the genes reported in Online Mendelian Inheritance in Man (OMIM), and the clinical value of these candidate genes was examined based on a literature search. Finally, the clinical data of the CAP patients were retrospectively analyzed. Detect the type of pathogenic bacteria in bronchial-alveolar lavage fluid (BALF) using metagenomics next-generation sequencing (mNGS) high throughput sequencing technology, and detect the expression of key genes through liquid based cell immunohistochemistry to analyze the correlation between pathogenic bacteria and key genes. Through the intersection of Venn diagrams, 175 co-expressed downregulated DEGs related to CAP were identified. A total of 4 candidate genes, including ICOS, IL7R, ITK, and ZAP70, were obtained by constructing the protein mutual aid network and conducting a module analysis of the common differentially expressed genes. The core genes in the GSEA enrichment pathways were intersected with the CAP-related genes reported in the relevant literature retrieved from the OMIM database. In the Venn diagram, two genes that coexist with OMIM include IL7R and PIK3R1. After considering our findings and the relevant literature, we determined that the key gene related to the occurrence and development of CAP was IL7R. The mNGS detected 13 kinds of bacteria, 4 kinds of fungi, and 2 kinds of viruses. Based on immunohistochemical results, it was found that there were relatively more bacteria detected in the IL7R high expression group. The identification of the key gene IL7R and the related signaling pathways extend understanding of the pathogenesis of CAP and provide a theoretical basis for clinical targeted therapy research.

Investigation of donor-derived Strongyloides stercoralis infection in multiple solid organ transplant recipients-California, Michigan, Ohio, 2022.

The Centers for Disease Control and Prevention led an investigation to determine if Strongyloides infection in a right kidney recipient was an existing chronic infection, or if the infection was transmitted from an infected organ donor. Evidence regarding the organ donor and organ recipients Strongyloides testing, treatment, and risk factors were gathered and evaluated. The case classification algorithm created by the Disease Transmission Advisory Committee was utilized. The organ donor had risk factors for Strongyloides infection; the banked donor specimen, submitted for serology testing 112 days post-donor death, was positive. The right kidney recipient was negative for Strongyloides infection pretransplant. Strongyloides infection was diagnosed via small bowel and stomach biopsies. The left kidney recipient had risk factors for Strongyloides infection. Two posttransplant Strongyloides antibody tests were negative at 59 and 116 days posttransplant; repeat antibody tests returned positive at 158 and 190 days posttransplant. Examination of bronchial alveolar lavage fluid collected 110 days posttransplant from the heart recipient showed a parasite morphologically consistent with Strongyloides species. She subsequently developed complications from Strongyloides infection, including hyperinfection syndrome and disseminated strongyloidiasis. Based on the evidence from our investigation, donor-derived strongyloidiasis was suspected in one recipient and proven in two recipients. The results of this investigation support the importance of preventing donor-derived Strongyloides infections by laboratory-based serology testing of solid organ donors. Donor positive testing results would direct the monitoring and treatment of recipients to avoid severe complications.

Serum Immunoglobulin G Testing against Pigeon Egg in Stable Fibrotic Hypersensitivity Pneumonitis

Introduction: The accuracy of serum immunoglobulin (Ig) G testing for diagnosis of stable bird-related fibrotic hypersensitivity pneumonitis (HP) is controversial. Furthermore, avian serum, extracts, or feathers were employed as antigens in bird-related HP; however, the usage of egg whites has not been reported. We investigated the utility of IgG testing against pigeon egg whites in patients with stable bird-related fibrotic HP. Methods: Patients having a positive inhalation test for pigeon antigen and a histological investigation with diagnostic confidence of fibrotic HP greater than moderate confidence were included. The control group consisted of patients with interstitial lung diseases (ILDs) other than HP. To select patients in the stable phase, patients with fibrotic HP were excluded if they were clinically considered to be in the acute exacerbation or acute phase. The IgG testing against pigeon egg whites by enzyme-linked immunosorbent assay and the commercialized anti-pigeon IgG testing by fluorescence enzyme immunoassay were investigated. Results: In this study, 37 patients with stable bird-related fibrotic HP and 32 patients with ILDs other than HP participated. Serum IgG testing for pigeon egg whites revealed that the control group’s optical density was 0.147 and the group with bird-related fibrotic HP had a mean value of 0.207 (p = 0.011). IgG testing in bronchial alveolar lavage fluid was not significantly higher in the bird-related fibrotic HP group than in controls (p = 0.42). No significant difference in area under the curve between an IgG testing against pigeon egg whites and a commercialized anti-pigeon IgG testing was observed (p = 0.24). Test accuracy for stable bird-related fibrotic HP ranged from 62% to 76% sensitivity and 59–66% specificity. Conclusion: IgG testing to identify the inciting antigen in patients with stable bird-related fibrotic HP had relatively low accuracy.

Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

Cigarette smoke (CS) is the leading cause of chronic obstructive pulmonary disease (COPD), which is characterized by chronic bronchial inflammation and emphysema. Growing evidence supports the hypothesis that dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is critically involved in the pathogenesis of CS-mediated COPD. However, the underlying mechanism remains unclear. Here, we report that supressed CFTR expression is strongly associated with abnormal phospholipid metabolism and increased pulmonary inflammation. In a CS-exposed mouse model with COPD-like symptoms, we found that pulmonary expression of sphingosine kinase 2 (SphK2) and sphingosine-1-phosphate (S1P) secretion were significantly upregulated. Therefore, we constructed a SphK2 gene knockout (SphK2-/-) mouse. After CS exposure for six months, histological lung section staining showed disorganized alveolar structure, increased pulmonary fibrosis, and emphysema-like symptoms in wild-type (WT) mice, which were less pronounced in SphK2-/- mice. Further, SphK2 deficiency also decreased CS-induced pulmonary inflammation, which was reflected by a remarkable reduction in pulmonary infiltration of CD45+CD11b+ neutrophils subpopulation and low levels of IL-6 and IL-33 in bronchial alveolar lavage fluid. However, treatment with S1P receptor agonist suppressed CFTR expression and increased Nf-κB-p65 expression and its nuclear translocation in CS-exposed SphK2-/-mice, which also aggravated small airways fibrosis and pulmonary inflammation. In contrast, inhibition of S1P signaling with the S1P receptor analogue FTY720 rescued CFTR expression, suppressed Nf-κB-p65 expression and nuclear translocation, and alleviated pulmonary fibrosis and inflammation after CS exposure. Our results demonstrate that SphK2-mediated S1P production plays a crucial role in the pathogenesis of CS-induced COPD-like disease by impairing CFTR activity and promoting pulmonary inflammation and fibrosis.

Sevoflurane alleviates lung injury and inflammatory response compared with propofol in a rat model of VV ECMO.

Although venovenous extracorporeal membrane oxygenation (VV ECMO) is a reasonable salvage treatment for acute respiratory distress syndrome (ARDS), it requires sedating the patient. Sevoflurane and propofol have pulmonary protective and immunomodulatory properties. This study aimed to compare the effectiveness of sevoflurane and propofol on rats with induced ARDS undergoing VV ECMO. Fifteen sprague-dawley (SD) rats were randomly divided into three groups: Con group, sevoflurane (Sevo) group and propofol (Pro) group. Arterial blood gas tests were performed at time pointsT0 (baseline), T1 (the time to ARDS), and T2 (weaning from ECMO). Oxygenation index (PaO2/FiO2) was calculated, and lung edema assessed by determining the lung wet:dry ratio. The protein concentration in bronchial alveolar lavage fluid (BALF) was determined by using bicinchoninic acid assay. Haematoxylin and eosin staining was used to evaluate the lung pathological scores in each group. IL-1β and TNF-α were also measured in the BALF, serum and lung. Oxygenation index showed improvement in the Sevo group versus Pro group. The wet:dry ratio was reduced in the Sevo group compared with propofol-treated rats. Lung pathological scores were substantially lower in the Sevo group versus the Pro group. Protein concentrations in the BALF and levels of IL-1β and TNF-α in the Sevo group were substantially lower versus Pro group. This study demonstrates that compared with propofol, sevoflurane was more efficacious in improving oxygenation and decreasing inflammatory response in rat models with ARDS subject to VV ECMO treatment.

Free-Living Amoeba Vermamoeba vermiformis Induces Allergic Airway Inflammation.

The high percentage of Vermamoeba was found in tap water in Korea. This study investigated whether Vermamoeba induced allergic airway inflammation in mice. We selected 2 free-living amoebas (FLAs) isolated from tap water, which included Korean FLA 5 (KFA5; Vermamoeba vermiformis) and 21 (an homolog of Acanthamoeba lugdunensis KA/E2). We axenically cultured KFA5 and KFA21. We applied approximately 1×106 to mice’s nasal passages 6 times and investigated their pathogenicity. The airway resistance value was significantly increased after KFA5 and KFA21 treatments. The eosinophil recruitment and goblet cell hyperplasia were concomitantly observed in bronchial alveolar lavage (BAL) fluid and lung tissue in mice infected with KFA5 and KFA21. These infections also activated the Th2-related interleukin 25, thymic stromal lymphopoietin, and thymus and activation-regulated chemokines gene expression in mouse lung epithelial cells. The CD4+ interleukin 4+ cell population was increased in the lung, and the secretion of Th2-, Th17-, and Th1-associated cytokines were upregulated during KFA5 and KFA21 infection in the spleen, lung-draining lymph nodes, and BAL fluid. The pathogenicity (allergenicity) of KFA5 and KFA21 might not have drastically changed during the long-term in vitro culture. Our results suggested that Vermamoeba could elicit allergic airway inflammation and may be an airway allergen.

TRAUMA-DERIVED EXTRACELLULAR VESICLES ARE SUFFICIENT TO INDUCE ENDOTHELIAL DYSFUNCTION AND COAGULOPATHY.

Although a number of studies have demonstrated increased release of extracellular vesicles (EVs) and changes in their origin differentials after trauma, the biologic significance of EVs is not well understood. We hypothesized that EVs released after trauma/hemorrhagic shock (HS) contribute to endotheliopathy and coagulopathy. To test this hypothesis, adoptive transfer experiments were performed to determine whether EVs derived from severely injured patients in shock were sufficient to induce endothelial dysfunction and coagulopathy. Methods: Total EVs were enriched from plasma of severely injured trauma/HS patients or minimally injured patients by ultracentrifugation and characterized for size and numbers. Under isoflurane anesthesia, noninjured naive C57BL/6J mice were administered EVs at varying concentrations and compared with mice receiving equal volume vehicle (phosphate-buffered saline (PBS)) or to mice receiving EVs from minimally injured patients. Thirty minutes after injection, mice were sacrificed, and blood was collected for thrombin generation (thrombin-antithrombin, thrombin-antithrombin complex [TAT] assay) and syndecan-1 by enzyme-linked immunoabsorbent assay (ELISA). Lungs were harvested for examination of histopathologic injury and costained with von Willebrand factor and fibrin to identify intravascular coagulation. Bronchial alveolar lavage fluid was aspirated from lungs for protein measurement as an indicator of the endothelial permeability. Data are presented as mean ± SD, P < 0.05 was considered significant, and t test was used. Results: An initial proof-of-concept experiment was performed in naive mice receiving EVs purified from severely injured trauma/HS patients (Injury Severity Score [ISS], 34 ± 7) at different concentrations (5 × 106 to 3.1 × 109/100 μL/mouse) and compared with PBS (control) mice. Neither TAT nor syndecan-1 levels were significantly different between groups at 30 minutes after EV infusion. However, lung vascular permeability and histopathologic injury were significantly higher in the EV group, and lung tissues demonstrated intravascular fibrin deposition. Based on these data, EVs from severely injured trauma/HS patients (ISS, 32 ± 6) or EVs from minimally injured patients (ISS, 8 ± 3) were administered to naive mice at higher concentrations (1 × 109 to 1 × 1010 EV/100 μL/mouse). Compared with mice receiving EVs from minimally injured patients, plasma TAT and syndecan-1 levels were significantly higher in the trauma/HS EV group. Similarly, bronchial alveolar lavage protein and lung histopathologic injury were higher in the trauma/HS EV group, and lung tissues demonstrated enhanced intravascular fibrin deposition. Conclusion: These data demonstrate that trauma/HS results in the systemic release of EVs, which are capable of inducing endotheliopathy as demonstrated by elevated syndecan-1 and increased permeability and coagulopathy as demonstrated by increased TAT and intravascular fibrin deposition. Targeting trauma-induced EVs may represent a novel therapeutic strategy.

Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia.

Background Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (e.g., insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii-mitochondrial large subunit ribosomal RNA.MethodsThe procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples.ResultsThis CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/µL transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia.ConclusionOur study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia.

Preexisting Trichinella spiralis infection attenuates the severity of Pseudomonas aeruginosa-induced pneumonia

BackgroundA range of helminth species involve the migration of developing larvae through the lung and establish chronic infections in the host that include potent immune regulatory effects. Trichinella spiralis is one of the most successful parasitic symbiotes. After released by intestinal female adult worms, newborn larvae of T. spiralis travel through the circulatory system to the lung and finally reach skeletal muscle cells. As unique inflammation modulator of intracellular parasitism, T. spiralis shows improved responses to autoimmune disease and viral pulmonary inflammation by exerting immunomodulatory effects on innate and adaptive immune cells.Methodology/Principal findingsC57BL/6 mice were divided into four groups: uninfected; helminth- T. spiralis infected; P. aeruginosa infected; and co-infected. Mice infected with T. spiralis were incubated for 6 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage fluid, blood and lung samples were analyzed. We found that T. spiralis induced Th2 response in the mouse lung tissue, increased lung CD4+ T cells, GATA3, IL-4, IL-5 and IL-13 expression. Pre-existing T. spiralis infection decreased lung neutrophil recruitment, inflammatory mediator IL-1β and IL-6 expression and chemokine CXCL1 and CXCL2 release during P. aeruginosa- pneumonia. Furthermore, T. spiralis co-infected mice exhibited significantly more eosinophils at 6 hours following P. aeruginosa infection, ameliorated pulmonary inflammation and improved survival in P. aeruginosa pneumonia.ConclusionsThese findings indicate that a prior infection with T. spiralis ameliorates experimental pulmonary inflammation and improves survival in P. aeruginosa pneumonia through a Th2-type response with eosinophils.