Journal: EMBO Molecular Medicine

Article Title: Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection

doi: 10.15252/emmm.201810233

Figure Lengend Snippet: A–C Detection of LL‐37 expression in the engineered mDASCs by immunofluorescence (A), real‐time quantitative PCR (B), and Western blot (C). Scale bar, 50 μm. BF, bright field. n = 10. Error bars, SEM. D Anti‐Krt5 (red) and anti‐P63 (green) immunostaining of WT‐ and LL‐37‐mDASC colonies. Scale bar, 70 μm. E Stem cell colony‐forming efficiency of WT‐ and LL‐37‐mDASCs during five serial passages. n = 6. Error bars, SD. F Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright‐field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm. G Co‐culture of bacteria with DASCs shows antimicrobial effects in dose‐dependent manner. Initial additions of PAO1 were 0.1 × , 0.5 × and 1 × 10 4 CFU, respectively. Co‐culture duration, 6 h. n = 4. Error bars, SEM. MOI, multiplicity of infection. H Co‐culture of bacteria with DASCs shows antimicrobial effects in time‐dependent manner. Initial concentration of PAO1 was 1 × 10 4 CFU. MOI = 1. n = 3. Error bars, SEM. I, J Preincubation of cells with anti‐LL‐37 antibody, but not IgG control, significantly reduced anti‐PAO1 (I) and anti‐ Escherichia coli (J) effects of LL‐37‐mDASCs. Initial dose of bacteria was 10 3 CFU. Co‐culture duration, 18 h. n = 4 in (I) and n = 3 in (J). Error bars, SEM. Data information: Statistics for graphs: unpaired two‐tailed t ‐test (B), two‐way ANOVA followed by Sidak's test (G, H) and one‐way ANOVA followed by Tukey's test (I, J). * P < 0.05; ** P < 0.01; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: 1 × 10 4 cells per well) within culture medium without antibiotics and FBS were pre‐incubated with 1 μg/ml anti‐LL‐37 antibody (HM2070, Hycult biotech) or mouse isotype antibody control (B30010M, Abmart) for 2 h, and then co‐cultured with P. aeruginosa or E. coli in a humidified CO 2 incubator.

Techniques: Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Immunostaining, Imaging, Co-Culture Assay, Infection, Concentration Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection

doi: 10.15252/emmm.201810233

Figure Lengend Snippet: A Cell growth curve of WT‐ and LL‐37‐mDASCs was measured by MTT assay. n = 3–5. Error bars, SD. B Soft agar assay of WT‐ and LL‐37‐mDASCs. Mouse melanoma cell line was included as a positive control. C Histogram showed that LL‐37‐mDASCs conditioned medium (CM) had potent growth inhibitory effect on PAO1. Initial addition of PAO1 was 1 × 10 4 CFU. n = 5. Error bars, SEM. D Histogram shows that LL‐37‐mDASCs CM had potent growth inhibitory effect on Escherichia coli . Initial addition of E. coli was 1 × 10 4 CFU. n = 3. Error bars, SEM. E Clone formation unit assay of E. coli following incubation with indicated cellular CM. F Preincubation of CM with anti‐LL‐37 antibody, but not mouse IgG, reduced the antimicrobial effect of LL‐37‐mDASCs against E. coli . n = 3. Error bars, SEM. Data information: Statistics for graphs: one‐way ANOVA followed by Tukey's test. * P < 0.05; *** P < 0.001.

Article Snippet: 1 × 10 4 cells per well) within culture medium without antibiotics and FBS were pre‐incubated with 1 μg/ml anti‐LL‐37 antibody (HM2070, Hycult biotech) or mouse isotype antibody control (B30010M, Abmart) for 2 h, and then co‐cultured with P. aeruginosa or E. coli in a humidified CO 2 incubator.

Techniques: MTT Assay, Soft Agar Assay, Positive Control, Incubation

Journal: Scientific Reports

Article Title: Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling

doi: 10.1038/s41598-018-27012-0

Figure Lengend Snippet: PALA treatment results in the secretion of broad-spectrum AMPs from NHDF that kill ESKAPE pathogens. ( a ) Conditioned supernatants from PALA treated NHDF reduce viability of three pathogens of the ESKAPE family. Triplicate mid-log phase cultures of MRSA, P. aeruginosa , and A. baumannii were supplemented with conditioned supernatants collected from NHDF treated with or without PALA (25 µM, 16 hours) and cfu determined after 2–4 hours. Each symbol shown represents mean values of an independent experiment performed in triplicate. Results representative of 6 independent experiments. Mean ± SD; significance determined by unpaired, 2-tailed, Students t-test; ***p < 0.001, ****p < 0.0001. ( b ) PALA treatment induces the secretion of AMPs from NHDF. AMP levels in conditioned media collected from triplicate cultures of NHDF treated with or without PALA (25 µM, 16 hours) were quantitated by ELISA. Results representative of 6 independent experiments performed. Mean ± SD; significance determined by unpaired, 2-tailed, Students t-test; *p < 0.05, **p < 0.01. ( c ) Transcription of the genes encoding HBD2 ( DEFB4A ), and cathelicidin ( CAMP ) is enhanced by PALA treatment, while transcripts of the gene encoding HBD3 ( DEFB103B ) are not increased. NHDF were treated with or without 25 µM PALA for 16 hours in triplicate and RNA extracted. Transcript levels relative to unstimulated cells were determined by qRT-PCR from three independent experiments. Mean ± SD; significance determined by 2-way ANOVA and Bonferroni multiple comparisons test; *p < 0.05, **p < 0.01.

Article Snippet: AMP levels in tissue homogenates was determined by ELISA for HBD2 (Peprotech, Rocky Hill, NJ), HBD3 (Peprotech), and cathelicidin (LL-37; Hycult, Plymouth Meeting, PA) according to manufacturer’s protocols.

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

Journal: Journal of Translational Medicine

Article Title: Conformational changes in myeloperoxidase induced by ubiquitin and NETs containing free ISG15 from systemic lupus erythematosus patients promote a pro-inflammatory cytokine response in CD4 + T cells

doi: 10.1186/s12967-020-02604-5

Figure Lengend Snippet: NETs from SLE patients are enriched in ISG15 and H2B is the main substrate. ISG15 and H2B expression was assessed in spontaneous and LPS induced NETs from SLE patients (n = 6) and healthy controls (n = 3) by indirect immunofluorescence (40X) and confocal microscopy. Spontaneous NETs from a representative SLE patient shows the presence of extracellular ISG15 in the NET ( a ). In contrast to the SLE sample, a representative healthy control image shows the absence of ISG15 in the NET ( b ). Blue (DNA), red (ISG15) and green (H2B). Cumulative data from SLE patients and healthy controls (n = 6 subjects per group) show increased expression of ISG15 in the SLE samples ( c ). In SLE samples, ISG15 and H2B colocalized inside NETs with a high Pearson (R = 0.81) and Costes p value (1.0) in a representative SLE patient ( d ) and boxes represent pooled data (n = 6 subjects per group) that show increased colocalization of SLE samples vs healthy controls ( e ). H2B with ISG15 had the higher colocalization R value compared to other NET proteins such as LL37 or HMGB1 (Additional file : Figures S5 and S6). * p < 0.05

Article Snippet: Additionally, we used mouse anti-human LL37 (LSBio™) or mouse anti-human HMGB1 ( Thermo Fisher™ ). (Additional file : Figures S5 and S6).

Techniques: Expressing, Immunofluorescence, Confocal Microscopy