Journal:

Article Title: Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis

doi: 10.1111/j.0959-9673.2003.00365.x

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Rabbit anti-human SP-D , , K. Reid (University of Oxford) , Donkey anti-rabbit F(ab′) 2 fragment , Jackson Immuno-research Laboratory , ( Holmskov 2000 ).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

doi: 10.3389/fphar.2022.1007527

Figure Lengend Snippet: Specificity of different modes of fusion. (A) 19,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) transfected with the luciferase and β-galactosidase reporter genes were pre-incubated with 10 μg/ml of anti-spike monoclonal antibody (ACROB SPD-M128), 75 μg/ml soybean trypsin inhibitor (SBTi), 10 μg/ml leupeptin or 10 μM of individual drugs before co-cultured with 19,000 donor 293T cells transfected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins (SARS-2-S) together with a plasmid encoding the T7 polymerase. The co-cultures were treated as indicated. Fusion activity was measured as luciferase activity normalized against β-galactosidase activity and expressed as a ratio to the solvent control in the respective cell type and fusion condition which is set as 1. Data are presented as mean+/-SD of >3 repeats. (B) Photomicrographs of fusion cell morphology. 50,000 target cells (293T-ACE2, 293T-ACE2-TMPRSS2) pre-incubated with 28 μg/ml anti-spike monoclonal antibody (Sino Biol, 40592-R001) or 10 μM of individual drugs were co-cultured with 50,000 donor 293T cells transfected with an empty vector or the SARS-CoV-2 spike protein. The co-cultures were treated as indicated. Cells were fixed and stained with methylene blue. Bright-field images are of the same magnification x100 and scale. Fields of entire or mostly syncytia formation are framed with red squares. Fields of entire or mostly intact cells are framed with orange squares. Scattered syncytia are circled or outlined red and labelled S. Scattered intact cells are circled or outlined orange and labelled C. Sporadic intact cells and syncytia are marked by orange and red arrowheads, respectively. (C) Viability was measured by XTT assays in fusion cells (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) under physiological pH and treated with compounds as in (A) . Data are expressed as % viability to solvent control which is set as 100%. Data are presented as mean+/-SD of three repeats. (D) Negative screen. One of the three cell types: 293T, 293T-ACE2 and 293T-ACE2-TMPRSS2 (30,000 cells) was co-transfected with plasmids encoding the T7 polymerase, luciferase and β-galactosidase reporter genes and then treated with compounds as in (A) . Luciferase, β-galactosidase and the luciferase/β-galactosidase ratio are presented as ratios to the solvent controls which are set as 1. Data are presented as mean+/-SD of three repeats * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Neutralizing antibodies were used at 10 μg/ml (ACROB SPD-M128) and 28 μg/ml (Sino Biol, 40592-R001), respectively.

Techniques: Transfection, Luciferase, Incubation, Cell Culture, Plasmid Preparation, Activity Assay, Staining

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: Bradford protein assay with corresponding bands from the Western blot. Column fraction protein concentrations were quantified using a Bradford assay. Protein was detected in 3 fractions eluted by gel filtration. In SPD, most of the protein (multimer) was collected in Fraction 3. In SPD-MAA, almost no multimer eluted while trimer (Fraction 7) and monomer (Fraction 9) were collected. Western blots for each slot blotted fraction were probed for SPD and SPD-MAA. The SPD column fractions showed the presence of SPD in 3 fractions. SPD-MAA column fractions only contained SPD in Fractions 7 and 9. Blots probed with anti-MAA detected protein from SPD-MAA column fractions 7 and 9 (not shown).

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Bradford Protein Assay, Western Blot, Bradford Assay, Filtration

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: Surfactant protein binding to S. pneumoniae . Plates were coated with bacteria and incubated with 0-20 µg/mL SPD, SPD-MAA, BSA (negative control), or BSA-MAA. Bound SPD was detected with antibodies by ELISA. Absorbance at 450 nm of bound SPD at the different concentrations was determined (n = 5), non-parametric Kruskal-Wallis test was performed, differences between SPD and SPD-MAA were significant at all concentrations (*p< 0.01). The data are the averages +/- SD of five experiments. The only significant difference between SPD-MAA and the BSA negative control was observed at 20 µg/mL. No binding was observed for BSA-MAA.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Protein Binding, Bacteria, Incubation, Negative Control, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: Aggregation of S. pneumoniae in the presence of surfactant protein. GFP-SP were incubated with 10 µg/ml SPD or SPD-MAA for 90 min in the presence of calcium (5 mM) and visualized by fluorescence microscopy (X 500). The average area of aggregated clumps of bacteria was measured per field of view. The data are the averages +/- SD of five experiments. In the presence of calcium, SPD significantly increased aggregation compared to SPD-MAA, *p<0.0003. No clumping or aggregation of pneumococci was observed in the absence of calcium.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Incubation, Fluorescence, Microscopy, Bacteria

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: MAA adduction of SPD decreases phagocytosis of S. pneumoniae . GFP- S. pneumoniae were incubated with the murine macrophage cell line RAW264.7. The percent of RAW264.7 cells containing internalized bacterial cells per field was determined using fluorescence microscopy (X 500) after quenching non-internalized GFP-SP with Trypan blue. The image results are a representative of 5 independent experiments summarized by graph. Bars represent averages +/- SD, *p<0.0004.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Incubation, Fluorescence, Microscopy

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: SPD-induced anti-bacterial permeability is decreased by MAA adduction. S. pneumoniae was incubated in the presence of either 10 µg/mL SPD or SPD-MAA for up to 30 min and supernatant media phosphatase release measured as a function of bacterial permeability. A significant reduction in permeability (*p<0.05) was observed between SPD and SPD-MAA. Bars represent averages +/- SD, n=5 experiments.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Permeability, Incubation

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: MAA adduction of SPD decreases cell viability in response to respiratory syncytial virus (RSV). Infection of BEAS-2B with RSV increases LDH release compared to control media (no RSV). SPD (10-100 µg/mL) significantly (p<0.0002) decreases RSV-induced LDH release vs. the absence of SPD. No such decrease is observed with either 10 µg/mL (*p<0.01) or 100 µg/mL (**p<0.006) SPD-MAA vs. equal amounts of SPD. Bars represent averages +/- SD, n=5 experiments.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Virus, Infection, Control

Journal: Frontiers in Immunology

Article Title: Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function

doi: 10.3389/fimmu.2022.866795

Figure Lengend Snippet: Summary model. Covalent modification of surfactant protein D (SPD) by malondialdehyde-acetaldehyde (MAA) adduction changes the structure of the protein from multimeric form to trimeric form and reduces anti-microbial innate defense.

Article Snippet: Secondary antibodies for SPD (HRP-conjugated rabbit-anti Goat; Invitrogen, Carlsbad, CA) and SPD-MAA (HRP-goat anti-rabbit; Rockland, Limerick, PA) were diluted 1:15,000 in Western blocking buffer with 3% BSA and incubated for 1 h at room temperature.

Techniques: Modification