bal fluid  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: A stem-loop RNA RIG-I agonist protects against acute and chronic SARS-CoV-2 infection in mice

doi: 10.1084/jem.20211818

Figure Lengend Snippet: SLR14 does not significantly elicit IFN-III responses in the respiratory tract. (A–C) Experimental scheme. K18-hACE2 mice were i.v. administered with 15 µg SLR14 or vehicle. 3 h after injection, BALF and lung tissues were collected for IFN-λ ELISA (B) and RT-qPCR (C), respectively. (D and E) Experimental scheme. Ifnar −/− mice were intratracheally administered with 10 11 genome copies of AAV9-hACE2 and allowed to rest for 2 wk before intranasal infection with 10 6 PFU SARS-CoV-2 (2019n-CoV/USA_WA1/2020). 15 µg SLR14 or vehicle were i.v. administered at 4 h after infection. Lung tissues were collected for virological analysis at 4 DPI. Measurement of vRNA at 4 DPI by RT-qPCR using the CDCN2 primer-probe set (E). Mean ± SEM; statistical significance was calculated by two-way ANOVA followed by Bonferroni correction (B and C) or one-way ANOVA followed by Tukey correction (E); ****, P ≤ 0.0001. Data are representative of two independent experiments.

Article Snippet: Concentration of IFN-λ in BAL fluid was determined by ELISA (DY1789B; R&D Systems) according to manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Infection

Journal: The Journal of Experimental Medicine

Article Title: A stem-loop RNA RIG-I agonist protects against acute and chronic SARS-CoV-2 infection in mice

doi: 10.1084/jem.20211818

Figure Lengend Snippet: SLR14-mediated disease prevention and antiviral control rely on IFN-I signaling. (A and B) Experimental scheme. K18-hACE2 mice were i.v. administered with 15 µg SLR14 or vehicle. 3 h after injection, BALF and lung tissues were collected for IFN-I ELISA (A) and RT-qPCR (B), respectively. (C–M) Experimental scheme. K18-hACE2 mice were intranasally infected with 10 3 PFU SARS-CoV-2 (2019n-CoV/USA_WA1/2020). 2 h before infection, 15 µg SLR14 or vehicle was i.v. administered. 24 h before SLR14 injection, half of the SLR14-treated mice were additionally given 2 mg anti-IFNAR antibodies. Weight loss and survival were monitored daily up to 14 DPI. In a separate cohort, lung and trachea tissues were collected for virological analysis 3, 6, and 8 DPI. Nasal washes and brain tissues were collected for virological analysis at 8 DPI. (C–E) Weight loss and survival of K18-hACE2 mice treated with vehicle + PBS, SLR14 + PBS, or SLR14 + αIFNAR from 1 to 14 DPI. (F–H) Measurement of vRNA in the lung parenchyma 3, 6, and 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (I–K) Measurement of vRNA in the trachea 3, 6, and 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (L and M) Measurement of vRNA in the nasal wash (L) or the brain (M) 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (N) The experimental scheme was similar to that of , with the exception that mice were infected with a sublethal dose of SARS-CoV-2. Sera were then collected from survivor mice 14 DPI and used for anti–SARS-CoV-2 S1 IgG measurement by ELISA. Mean ± SEM; statistical significance was calculated by two-way ANOVA followed by Bonferroni correction (A and B), log-rank Mantel–Cox test (E), or one-way ANOVA followed by Tukey correction (F–M); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are pooled from or representative of two independent experiments.

Article Snippet: Concentration of IFN-λ in BAL fluid was determined by ELISA (DY1789B; R&D Systems) according to manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Infection

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma

doi: 10.3390/ijms24054719

Figure Lengend Snippet: OATD-01 ameliorates TGFβ1, IL-13, and CHIT1 in chronic HDM model in a dose-dependent fashion. ( A ) mRNA expression of Il13 in the lungs and ( B ) active TGFβ1 levels in BAL fluid in mice with chronic HDM (7 week) treated with OATD-01 (3 and 30 mg/kg; PO; single dose) or vehicle control. ( C ) Number of macrophages in BAL fluid in mice with chronic HDM (7-week-long) treated with OATD-01 (3 and 30 mg/kg; PO; single dose) or vehicle control. The level of CHIT1 protein in BAL fluid of control and HDM-instilled mice treated with vehicle or OATD-01 (30 mg/kg) in chronic airway inflammation model as evaluated by ( D ) Western blot and ( E ) corresponding densitometry analysis. Data presented as mean ± s.e.m. p -values < 0.05 were considered as statistically significant and presented as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Article Snippet: For quantification of active TGFβ1 level in BAL fluid ELISA (cat. No. 437707, eBioscience, San Diego, CA, USA) was used according to manufacturer’s protocol.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma

doi: 10.3390/ijms24054719

Figure Lengend Snippet: Inhibition of macrophage-specific CHIT1 attenuates airway remodeling. Decreased macrophage-specific CHIT1 expression and activity changes phenotype of profibrotic macrophages and blocks TGFβ1 and subsequent Th2-dependent IL-13 production. OATD-01 inhibits CHIT1-dependent macrophage–fibroblast positive feedback loop leading to fibroblast activation and myofibroblast transition. The overall effect of CHIT1 inhibition results in less collagen deposition in extracellular space, decreased goblet cell hyperplasia, and attenuated airway remodeling—features associated with severe asthma.

Article Snippet: For quantification of active TGFβ1 level in BAL fluid ELISA (cat. No. 437707, eBioscience, San Diego, CA, USA) was used according to manufacturer’s protocol.

Techniques: Inhibition, Expressing, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Spatial expression of an mRNA encoding Tie2-agonist in the capillary endothelium of the lung prevents pulmonary vascular leakage

doi: 10.1101/2022.10.12.511878

Figure Lengend Snippet: (A) Schematic diagram of the experimental protocol of the ex vivo perfused and ventilated mouse lung model. 6 h, 15 h after administration of mRNA-76-cLNP (1, 1.5 and 2 mg/kg) or control mRNA LUC -LNP (2 mg/kg) lungs were isolated and stimulated with PLY (1.4 μg/ml) for 1 minute. After 30 min, lung vascular permeability was assessed by quantifying respective concentrations of continuously infused human serum albumin (HSA) in the bronchoalveolar lavage fluid (BALF). (B) Graphic showing treatment with mRNA-76 to significantly decrease pneumolysin-induced hyperpermeability of mouse lungs as compared to treatment with control mRNA LUC -LNP 6 h (left) and 15 h (right) post mRNA-76-cLNP treatment as shown by HAS ELISA. Values are given as mean ( n = 10, **p<0.01 between indicated groups) concentration of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). HSA concentration of individual mice are indicated as dots.

Article Snippet: Thirty minutes after PLY challenge, bronchoalveolar lavage (BAL) was performed (2 x 650 μl, 0.9 % saline buffer), and HSA concentration was measured in BAL fluid (BALF) via ELISA (Bethyl Laboratories, cat. #E88-129) in order to quantify the lung barrier failure ( ; ).

Techniques: Ex Vivo, Control, Isolation, Permeability, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: bioRxiv

Article Title: Spatial expression of an mRNA encoding Tie2-agonist in the capillary endothelium of the lung prevents pulmonary vascular leakage

doi: 10.1101/2022.10.12.511878

Figure Lengend Snippet: (A) Schematic drawing of the experimental protocol. Animals (n=10) were challenged intratracheally with 0.9% w/v saline or LPS (3 mg/kg). A fixed volume of 50 μL, equal to an approximate intratracheal dose volume of 2.5 mL/kg, based on a 20 g mouse, was applied intratracheally. 2h after LPS administration mRNA-76-cLNP (1.5 mg/kg IV) or mRNALUC-cLNP01 (1.5 mg/kg IV) was intravenously administered, and bronchoalveolar lavage fluid (BALF) was analysed after 24 h. (B) Effect of COMP-Ang1 expression on BALF total and differential cell counts and on wet lung weight in a murine model of endotoxin (LPS)-induced pulmonary inflammation. Data shown as mean ± standard error of the mean. *** p <0.001 when compared to saline challenged control group. # p <0.05, ## p <0.01, ### p <0.001 as compared to LPS challenged and vehicle treated group.

Article Snippet: Thirty minutes after PLY challenge, bronchoalveolar lavage (BAL) was performed (2 x 650 μl, 0.9 % saline buffer), and HSA concentration was measured in BAL fluid (BALF) via ELISA (Bethyl Laboratories, cat. #E88-129) in order to quantify the lung barrier failure ( ; ).

Techniques: Saline, Expressing, Control

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Ezrin expression and epithelial cell–cell adhesion were decreased in an ovalbumin (OVA)-treated allergic mouse model of asthma and restored by anti–IL-13 treatment. (A) Hematoxylin and eosin (H&E) staining of lung tissue in “asthma mice” (black arrows indicate bronchial epithelial cells). Representative image of E-cadherin and ZO-1 immunostaining (black arrows in the middle and bottom panels indicate their expression on the bronchial epithelial cells) was examined in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and was analyzed by Image-Pro Plus 6.0. Scale bars, 50 μm. (B) Epithelial cell–cell adherence was determined by electron microscopy (scale bars, 1 μm; white arrow). (C) Immunohistochemical analysis of ezrin expression in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13) (original magnification, ×400; scale bar = 100 μm; black arrow) and scored (right graph). (D) The concentrations of ezrin in BAL fluid (BALF) of OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and IL-13 of asthma mice and controls were measured using an ELISA. The data are presented as mean ± SEM and were analyzed by Student’s t test (control group, n = 8–15; asthma group; n = 8–17). The correlation between ezrin and IL-13 in BALF of mice was analyzed by Pearson’s correlation test. ns = not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with respective controls.

Article Snippet: The levels of IL-4, IL-5, IL-13 (R&D Systems), and ezrin in mouse BAL fluid (BALF; CSB-EL007914MO; Cusabio) and human serum ezrin (SEB297Hu; Cloud Clone Corp.), IL-13, periostin, and IgE were measured by ELISA kit, according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Immunostaining, Saline, Control, Electron Microscopy, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay