Journal: JBMR Plus

Article Title: Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice

doi: 10.1093/jbmrpl/ziad005

Figure Lengend Snippet: HuBRGSF mice infected with isogenic PVL mutant had fewer human myeloid cells and dead cells in the bone niche. Myeloid cell populations in bone marrow of huBRGSF mice with a transtibial implant-associated osteomyelitis after infection with MRSA USA300 WT, USA300 Δpvl, or USA300 Δpvl+pvl. Percentages of (A) human CD45 + myeloid cells, (B) neutrophils, (C) monocytes/macrophages, (D) dendritic cells, (E) NK cells, (F) HLA-DR + neutrophils, (G) HLA-DR + monocytes/macrophages, or (H) eFluor780 + dead cells in bone marrow of WT- (circle), Δpvl- (square), or Δpvl+pvl- (triangle) infected mice, 3 d after surgery are depicted. Data are mean ± SD and was analyzed with a Tukey’s multiple comparison test of a one-way ANOVA. N = 6 for WT-infected mice, n = 5 for Δpvl-infected mice, and n = 6 for Δpvl+pvl-infected mice. * p < 0.05, * * p < 0.01, * * * p < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescent staining of HuBRGSF infected limbs (USA300 WT, USA300 Δpvl, or USA300 Δpvl+pvl): mouse anti-CEACAM8/CD66b for neutrophils (Clone G10F5, NBP2–80664, Novus Biologicals, Zug, Switzerland), rabbit anti- S. aureus (PA1–7246, RRID:AB_561546, Thermo Fisher Scientific), and mouse anti-CD68 for monocytes (Clone PG-M1, MS-1808-S1, RRID:AB_149350, Thermo Fisher Scientific).

Techniques: Infection, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: Exosomes were isolated from plasma of 17 healthy donor controls and 17 PAH patients, using the ExoTIC device described under ‘Methods’. CD66b positive neutrophil exosomes were pulled down using anti-CD66b beads, from pooled exosomes of Con and PAH pooled plasma. (a) Size distribution of the pooled exosomes, determined using Nanosight. (b) Representative transmission electron microscopy (TEM) images of CD66b positive neutrophil exosomes derived from pooled plasma of PAH vs. Con patients. Scale bar=100 nm (c) Western immunoblot analysis and quantification of NE and HERV-K envelope from pooled PAH vs. Con neutrophil exosomes, relative to the exosome marker CD9. H3 from PAH neutrophil total lysate was used as a negative control. (d) NE activity in PAH vs. Con exosomes after 120 min incubation. NE was assessed by the production of BODIPY FL labeled fluorescent elastin fragments from self-quenching BODIPY FL-conjugated bovine neck ligament elastin. Bars represent mean ± SEM n=3 technical replicates of the pooled exosomes. *p<0.05, **p<0.01 by unpaired Student t-test.

Article Snippet: We therefore used a CD66b antibody to purify neutrophil specific exosomes from PAH and control plasma after total exosome isolation using a nanofiltration-based exosome isolation tool, Exosome Total Isolation Chip (ExoTIC) .

Techniques: Isolation, Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Marker, Negative Control, Activity Assay, Incubation, Labeling

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: CD66b exosomes were isolated from plasma of PAH patients or healthy controls. Adult male mice (8 weeks) were injected with PAH vs. Con plasma exosomes (1.63 × 10 7 ) at a volume of 100 µL. The PAH plasma exosomes were preincubated for 30 min with either elafin (0.02 mg/kg mouse weight in a volume of 4-6 µL from a 100 µg/mL stock solution), and or an equivalent volume of vehicle (PBS). The exosome suspensions were then injected twice a week, for 5 weeks. Hemodynamic function was evaluated 2 days after the last injection. (a) Illustration of animal protocol. (b) Pulmonary artery acceleration time (PAAT). (c) Right ventricular systolic pressure (RVSP). (d) Right ventricular hypertrophy (RVH). (e) Microscopy images of lung sections of the mice, labeled for αSMA (brown, smooth muscle cell marker) showing examples of nonmuscular, partially muscular and fully musularized vessels. Scale bar=40 µm. In b-e bars represent mean ± SEM. *p<0.05 **p<0.01, ***p<0.001 by one-way ANOVA followed by Dunnet’s post test comparing each group mean with the control group.

Article Snippet: We therefore used a CD66b antibody to purify neutrophil specific exosomes from PAH and control plasma after total exosome isolation using a nanofiltration-based exosome isolation tool, Exosome Total Isolation Chip (ExoTIC) .

Techniques: Isolation, Injection, Microscopy, Labeling, Marker

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software