recombinant ace2 race2  (R&D Systems)

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: Significant increase of CR Abs IgG in moderate/severe COVID-19 patients’ sera may contribute to cytokine secretion and NET formation in human leukocytes in the presence of RBD. IgG against nucleocapsid, N, RBD and ACE2 proteins in sera from COVID-19 patients and healthy donors (HD) were measured by ELISA. Comparison of ( A ) the levels of IgG against N, ( B ) RBD, and ( C ) ACE2 in mild vs. moderate/severe COVID-19 patients. ( D ) Anti-ACE2 IgG levels in sera of mild vs. moderate/severe COVID-19 patients after BSA or RBD preadsorption. For the leukocyte activation test, isolated human leukocytes were preincubated with 1:100 diluted HD serum or sera from CR Ab-positive COVID-19 patients (CR serum) as indicated in the presence or absence of additional rACE2 (10 μg/mL) for 30 min. Afterward, unbound antibodies were removed by centrifugation, and the cells were treated with the RBD protein (10 μg/mL) for 24 h. The supernatants were collected to measure the ( E ) TNF-α, ( F ) IL-1β, ( G ) IL-6, ( H ) IL-8, and ( I ) MPO levels using ELISA kits. The averages of triplicate cultures ± SD are shown. Multiple comparison of antibody response and leukocyte activation tests ( n = 3–5) was conducted by one-way ANOVA and Tukey’s post hoc test. The comparison between BSA and RBD preadsorption was analyzed by paired Student’s t-test; P values were displayed, and for values less than 0.0001, they were represented as ****

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Activation Assay, Isolation, Centrifugation

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: CR Abs induce human neutrophils to secrete IL-8 and MPO in the presence of RBD. ( A , B ) Isolated human neutrophils (2 × 10 6 cells/mL, 0.3 mL/test) were treated with the recombinant RBD protein in the presence or absence of the indicated antibodies (mAb 127 or cmIgG) for different time points (1, 3, 6, and 24 h). ( C , D ) Neutrophils were cotreated with 10 μg/mL RBD recombinant protein and mAb 127 or cmIgG at different concentrations (2.5, 5, or 10 μg/mL). ( E , F ) Neutrophils were stimulated with the recombinant RBD protein in the presence or absence of the indicated antibodies (mAbs 127, LGSV201, cmIgG, or anti-ACE2) for 24 h. The supernatants from these experiments were collected, and IL-8 and MPO levels were quantified using ELISA kits. The averages of triplicate cultures ± SD are shown. Statistical significance was calculated using one-way ANOVA and Tukey’s post hoc test; P values were displayed, and for values less than 0.0001, they were represented as ****

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Isolation, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: NETosis triggered by CR Abs in the presence of RBD drives thrombosis-associated cell activation. ( A ) Isolated human neutrophils were treated with 10 μg/mL recombinant RBD protein and the indicated antibodies (mAbs 127, LGSV201, or cmIgG, 10 μg/mL) or not. After the indicated time points, the cell suspensions were spun onto a microscope slide by using a cytocentrifuge, fixed and stained with anti-CD66b antibody (green), anti-MPO antibody (red) and DAPI (blue) nuclear stain and then visualized using immunofluorescence staining. The area of NET formation at ( B ) different time points or ( C ) 24 h after stimulation was quantified by ImageJ. Views for NET quantification were randomly selected with 9 pictures from each experiment. The supernatants of neutrophils were harvested after different treatments and further administered to isolated human PBMCs, HUVECs, or platelets. Purified PMA-induced NETs (1 μg/mL) were used as the positive control, and in some experiments, rACE2 was added. Following 24 h of stimulation with neutrophil-conditioned media, PBMC supernatants were collected to measure the ( D ) TNF-α and ( E ) IL-6 levels using ELISA kits. ( F ) Endothelial barrier integrity was determined by a Transwell permeability assay, as described in the Materials and Methods. ( G ) After 15 min of stimulation, the percent fluorescence of P-selectin surface expression on platelets was measured by anti-CD62p FITC-conjugated antibodies and further analyzed by cytoFLEX. The averages of triplicate cultures ± SD are shown. Statistical significance was calculated using one-way ANOVA and Tukey’s post hoc test, **** p < 0.0001. Bar: 25 μm

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Activation Assay, Isolation, Recombinant, Microscopy, Staining, Immunofluorescence, Purification, Positive Control, Enzyme-linked Immunosorbent Assay, Permeability, Fluorescence, Expressing

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: CR Abs-triggered NETosis is dependent on ACE2 and the Fc receptor. Isolated human neutrophils were treated with recombinant RBD protein (10 μg/mL) and the indicated antibodies (mAb 127, mAb 127-F(ab’) 2 , or cmIgG-F(ab’) 2 , 10 μg/mL) in the presence or absence of additional rACE2 (1 or 10 μg/mL) for 24 h. The supernatants were collected to measure the levels of ( A , E ) IL-8 and ( B , F ) MPO using ELISA kits. ( C , D , G , H ) The cell suspensions were spun onto a microscope slide by using a cytocentrifuge and were fixed and subjected to immunofluorescence staining with an anti-MPO antibody (green), anti-histone antibody (red) and Hoechst (blue) nuclear stain, and the area of NET formation was quantified by ImageJ. The averages of triplicate cultures ± SD are shown. The views for NET quantification were randomly selected with 9 pictures from each experiment. Statistical significance was calculated using one-way ANOVA and Tukey’s post hoc test; P values were displayed, and for values less than 0.0001, they were represented as ****. Bar: 25 μm

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Isolation, Recombinant, Enzyme-linked Immunosorbent Assay, Microscopy, Immunofluorescence, Staining

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: CR IgG purified from unvaccinated COVID-19 patients induces neutrophil activation and NETosis in the presence of RBD. Isolated human neutrophils were preincubated with 100 μg/mL purified IgG from HD serum (HD IgG) or different COVID-19 patient sera: anti-RBD IgG (anti-ACE2 antibody negative but anti-RBD antibody positive), anti-ACE2 IgG (anti-ACE2 antibody positive), or CR IgG (CR antibody positive) in the presence or absence of additional rACE2 (10 μg/mL) or dasatinib (50 nM) as indicated for 30 min. After removing unbound antibodies by centrifugation, cells were treated with the RBD protein (10 μg/mL) for 24 h. ( A ) IL-8 and ( B ) MPO levels in the supernatants were measured using ELISA kits. ( C , D ) Cell suspensions were cytocentrifuged onto microscope slides, fixed, and subjected to immunofluorescence staining with anti-MPO antibody (green), anti-histone antibody (red), and Hoechst nuclear stain (blue). The red arrow indicates neutrophils that released NETs. The area of NET formation was quantified using ImageJ. Averages of triplicate cultures ± SD are shown. Views for NET quantification were randomly selected with 9 pictures from each experiment. Statistical significance was assessed using one-way ANOVA and Tukey’s post hoc test; **** p < 0.0001. Bar: 25 μm

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Purification, Activation Assay, Isolation, Centrifugation, Enzyme-linked Immunosorbent Assay, Microscopy, Immunofluorescence, Staining

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: Distribution of disease severity, anti-ACE2 autoantibody and CR antibody positive rate in COVID-19 patients with vaccination (Vac) or without (UnVac)

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques:

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: Vaccination is conducive to decreasing CR Abs IgG levels in the sera of COVID-19 patients. IgG against ( A ) RBD, ( B ) nucleocapsid, N and ( C ) ACE2 proteins in sera from different groups were measured by ELISA. ( D ) Anti-ACE2 IgG levels in sera of unvaccinated COVID-19 patients and vaccinated COVID-19 patients after BSA or RBD preadsorption. Multiple comparisons in COVID-19 samples were conducted by two-way ANOVA. The comparison between BSA and RBD preadsorption was analyzed by paired Student’s t-test; P values were displayed, and for values less than 0.0001, they were represented as ****

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19

doi: 10.1186/s12929-024-01026-5

Figure Lengend Snippet: The potential mechanisms underlying CR Ab-induced thrombosis. (1) CR Abs binding to RBD and ACE2 as well as Fc receptors on the surface of neutrophils induce signaling pathways. (2) Signaling pathways that regulate IL-8 secretion and NET formation are activated: SFK-AMPK signaling mediates IL-8 secretion, while SFK-PI3K-PAD4 signaling mediates NET formation. The actions of both IL-8 and NETs could be suppressed by dasatinib, which is an SFK inhibitor. (3) Secreted IL-8 and NETs subsequently cause (4) the secretion of TNFα, IL-6, and IL-1β from PBMCs, (5) enhanced expression of surface P-selectin on platelets, and (6) increased endothelial permeability. These processes collectively contribute to the eventual formation of thrombi. CR Ab, ACE2-cross-reactive RBD antibodies; ACE2, angiotensin converting enzyme-2; RBD, SARS-CoV-2 receptor binding domain; IL-8, interleukin-8; NETs, neutrophil extracellular traps; SFK, Src family kinases; AMPK, AMP-activated protein kinase; PI3K, phosphoinositide 3-kinase; PAD4, protein arginine deiminase 4; TNFα, tumor necrosis factor alpha; IL-6, interleukin-6; IL-1β, interleukin-1β; PBMC, peripheral blood mononuclear cells. Created with BioRender.com

Article Snippet: Recombinant human ACE2 (rACE2) was purchased from Genetex (Irvine, CA).

Techniques: Binding Assay, Protein-Protein interactions, Expressing, Permeability

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: SARS-CoV-2 infection in male and female k18-hace2 mice over 7 days post-infection (dpi) (A) Body weight loss in response to intranasal inoculation of SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse). ∗p < 0.05 vs. male. Data represented in median ± IQR. (B) Lung injury scoring assessed on a scale of 0–4 for each of the following criteria: 1) neutrophil numbers in the alveolar space, 2) alveolar septal thickening, 3) number of hyaline membranes, 4) alveolar hemorrhage, and 5) cellular hyperplasia. ∗p < 0.05 vs. Ctrl and male. Data represented in mean ± SEM. (C) Viral RNA levels in oropharyngeal swabs at 2, 4, and 6 dpi. ∗p < 0.05 vs. male. Data represented in median ± IQR. (D–G) Copy numbers of E-gene and RdRp-gene in multiple organ tissues. ∗p < 0.05 vs. Ctrl. Data represented in mean ± SEM. (H) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (I) Protein expression in lung tissue detected by western blots in vehicle control (C) and at 7 dpi. (J) Concentration of soluble RAGE protein in plasma in vehicle control and at 7 dpi. ∗p < 0.05 vs. other groups. Data represented in mean ± SEM. (K) Correlation of protein levels of ACE2 and ERα in lung tissue of male mice before and after infection at 7 dpi. (L) Estrogen receptor alpha (ERα)/androgen receptor (AR) ratio assessed from western blot analysis in relation to β-actin loading control. ∗p < 0.05 vs. male. Data represented in mean ± SEM. (M) Concentration of 17β Estradiol in lung tissue homogenates in vehicle control and at 7 dpi. ∗p < 0.05 vs. male. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S1 ).

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Infection, Expressing, Western Blot, Control, Concentration Assay, Clinical Proteomics

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Inhalation of rACE2 attenuates SARS-CoV-2 infection, replication, and lung injury and recovers expression of endogenous ACE2 and ERα in K18-hACE2 male mice at 7 dpi (A) Study scheme. Mice were intranasally inoculated with SARS-CoV-2 (4 × 10 5 TCID50/50μL/mouse) and 48 h later received daily nebulization of rACE2 (12 mg/kg) or vehicle control solution for 5 days. (B and C) Copy numbers of viral envelope (E) gene by RT-qPCR and titers (TCID50) in lung tissue in the SARS-CoV-2 infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (D) Gene expression of IL-6 in lung tissue in the infection alone and rACE2-treated groups. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (E) Concentration of soluble RAGE protein in plasma in infection alone and rACE2-treated mice. ∗p < 0.05 vs. SARS-CoV-2. Data represented in mean ± SEM. (F) Detection of SARS-CoV-2 nucleocapsid protein (red) in lung tissues. Scale bars, 50 μm (main images) and 20 μm (magnified). (G) Representative lung histology by H&E staining. Arrows indicate neutrophil infiltration in the alveolar space. Lung injury scores from 6 animals per group are also shown. ∗p < 0.05 vs. naive control (C), ♰p < 0.05 vs. SARS-CoV-2 alone & (C). Data represented in mean ± SEM. (H and I) Protein expression in lung tissue detected by western blots in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C & rACE2-treated groups. Data represented in mean ± SEM. (J and K). Fold changes in the concentrations of 17β Estradiol and ERα/AR ratio assessed from western blot analysis in relation to β-actin loading control in lung tissue in naive control (C), SARS-CoV-2 (CoV2) alone, and rACE2-treated groups. ∗p < 0.05 vs. C and SARS-CoV-2, respectively. n = 5–7 biologically independent mice for all groups. Data represented in mean ± SEM. (see also Figure S3 ).

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Infection, Expressing, Control, Quantitative RT-PCR, Gene Expression, Concentration Assay, Clinical Proteomics, Staining, Western Blot

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Nebulization of rACE2 partially restores the ERα-associated signaling proteomic profiles in lung tissue at 7 dpi Proteomic analysis revealed that treatment with rACE2 resulted in an increase in 31 ERα-associated proteins and a decrease in 10 proteins. Of note, 16 of these proteins (highlighted in purple) were also found in female mice without rACE2 treatment at 7 dpi.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques:

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Treatment with rACE2 attenuates Delta variant replication and viral load and decreases cytopathic effects in human lung organoids (A) Human lung organoids expressing pro-surfactant protein C (SPC) and epithelial cell adhesion molecule (EpCAM). Scale bars, 20 μm (main images). (B) Representative images of human lung organoids in naive control (Ctrl, upper panel), Delta variant-challenged (middle panel), and treated with a pre-mixture of rACE2 (400 μg/mL) and Delta variant (1.8 × 10 7 TCID50/mL) for 30 min (lower panel). The expression of ACE2 (red) and viral nucleocapsid protein (green) were detected at 4 h post-infection. Scale bars, 5 μm. (see also Figure S4 ). (C) Levels of viral envelope (E) gene RNA were assessed by qRT-PCR after treatment with various concentrations of rACE2 in human lung organoids at 3 dpi. Supernatants of the Delta variant-infected lung organoids were collected and used to infect Vero E6 cells for TCID50 assay. ∗p < 0.05 vs. Mock; ♰ p < 0.05 vs. Delta variant alone. Data represented in mean ± SEM. (D) Representative images of cytopathic effects of human lung organoids in Delta variant infection and rACE2-treated groups at 3 dpi. Scale bars, 1000 μm (main images) and 400 μm (magnified). Data presented are representatives of three independent experiments.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Variant Assay, Expressing, Control, Infection, Quantitative RT-PCR, TCID50 Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet: Proposed mechanisms underlying biological sex differences in outcomes of SARS-CoV-2 and Delta variant infections and the therapeutic potential of inhaled ACE2 (A) Under physiological conditions, ACE2 catalyzes the conversion of Ang II to Ang1-7 for organ protection. The ACE2 gene is located at X chromosome p22.2, where genes are known to escape X-inactivation, contributing to phenotypic differences of ACE2 between biological sexes. Membrane expression of ACE2 can be upregulated by estrogens through estrogen receptor α (ERα), which exerts anti-inflammatory properties and further enhances organ protection. In contrast, androgen receptor (AR) can cleave ACE2 and mediate pro-inflammation and tissue injury. . (B) During SARS-CoV-2 infection, the virus binds to and decreases host cell surface ACE2. For a given infection, the reduction of endogenous ACE2 expression is associated with a dramatic decrease in ERα expression, resulting in loss of organ protection in males. In contrast, expression of endogenous ACE2 was sustained in females, potentially due to the extra X-linked ACE2 and well-maintained ERα expression, leading to compensatory mechanisms and persistent organ protection. (C) Inhalation of rACE2 can restore the balance of endogenous ACE2 and ERα by blocking or attenuating SARS-CoV-2 from binding to membrane ACE2 in males.

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Variant Assay, Membrane, Expressing, Infection, Virus, Blocking Assay, Binding Assay

Journal: iScience

Article Title: Inhalation of ACE2 as a therapeutic target on sex-bias differences in SARS-CoV-2 infection and variant of concern

doi: 10.1016/j.isci.2023.107470

Figure Lengend Snippet:

Article Snippet: Male K18-hACE2 mice aged 8–10 weeks received soluble human recombinant ACE2 (rACE2, Apeiron Biologics, Vienna, Austria) either by intravenous injection (0.4 mg/kg) or nebulization (DD 12 mg/kg).

Techniques: Virus, Isolation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software