Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) After mixing with MRSA (1.2 × 106 CFU/ml), blood samples isolated from mice were assessed for their ability to eliminate MRSA after a 2- or 5-hour incubation. (B) Ex vivo killing of K. pneumoniae (1.54 × 106 CFU/ml) by whole blood isolated from ACEI- or ARB-treated mice was evaluated. (C) Ex vivo killing of P. aeruginosa (1.9 × 106 CFU/ml) by whole blood isolated from ACEI- or ARB-treated mice was evaluated. (D) In vitro intracellular killing of MRSA by purified murine neutrophils from bone marrow at 2 and 5 hours was evaluated. (E) In vitro intracellular killing of K. pneumoniae by purified neutrophils from mice treated with ACEIs or an ARB is shown. (F) In vitro intracellular killing of P. aeruginosa by purified neutrophils from mice treated with ACEIs or an ARB is shown. In the intracellular killing assays (D to F), neutrophils were isolated from bone marrow and infected with the indicated bacterial strains at an MOI of about 15. A one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM (n = 6). **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, nonsignificant.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Isolation, Incubation, Ex Vivo, In Vitro, Purification, Infection

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) WT and ACE KO mice were infected with MRSA intravenously (8 × 107 CFU/100 μl). The bacterial concentration in the blood is shown at 24 hours after infection. (B to D) Bacterial count in spleen (B), liver (C), and lung (D) was determined at 48 hours after infection. Indicated groups of mice were treated with either ramipril (Ram; 40 mg/liter) or losartan (Los; 600 mg/liter) for 1 week before infection. Drugs were administered throughout the study. Neutrophil depletion (Dep) was achieved by daily injection of mice with a murine anti-PMN antibody, starting 24 hours before infection with MRSA to the end of study. Mice were perfused with PBS before harvesting tissues. Two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM (n = 7 mice per group). ***P < 0.001 and ****P < 0.0001.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Infection, Concentration Assay, Injection

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) Neutrophils isolated from ACE KO, WT, and NeuACE mice were stimulated with 1 μM LPS for 30 min followed by staining with Apopxin dye staining and flow cytometry to measure apoptosis. To inhibit LTB4, mice were treated with zileuton (5 mg/kg per day, gavage) for 2 days before isolation of bone marrow neutrophils, and then, cells were treated with 100 μM zileuton during the in vitro assay (n = 5 mice per group). (B) Western blots showing the presence of phospho-PI3K p85α, phospho-AKT, and phospho–NF-κB p65 are shown. (C) Western blots showing the presence of apoptotic markers MCL-1 and BAD. (D) Western blots show the abundance of phospho-PI3K p85α, phospho-AKT, phospho–NF-κB p65, Mcl-1, and BAD in neutrophils isolated from mice treated with ramipril. Mice were treated with ramipril (40 mg/liter) in drinking water for 7 days before bone marrow neutrophil isolation, and then, 10 μM ramipril was added in vitro. (E) Western blots show the abundance of phospho-PI3K p85α, phospho-AKT, phospho–NF-κB p65, Mcl-1, and BAD in neutrophils isolated from mice treated with zileuton. To inhibit LTB4 synthesis, mice were treated with zileuton (5 mg/kg per day) in drinking water for 2 days before neutrophil isolation. Zileuton was maintained at a concentration of 100 μM in vitro. For (B) to (E), neutrophils were treated with 1 μM LPS for 30 min before being lysed, and β-actin serves as the loading control. Numbers below bands indicate relative band density as determined by Image Studio Lite version 5.2 (LI-COR). A two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. *P < 0.05.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Isolation, Staining, Flow Cytometry, In Vitro, Western Blot, Concentration Assay, Control

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) Representative images of hematoxylin and eosin–stained murine heart transverse sections with or without aortic valve damage by catheter insertion are shown. The blue arrow indicates aortic valve damage and infiltration of cells. Scale bars, 100 μm. (B) Bacteria were counted per heart in ACE KO, WT, and NeuACE mice challenged with intravenous MRSA (about 1 × 107 CFU/100 μl) without valve damage. (C) Bacteria were counted per heart in mice challenged with intravenous MRSA with aortic valve injury. ACE KO, WT, and NeuACE mice were untreated, depleted of neutrophils, or treated with ramipril or losartan. n ≥ 7. (D) Incidence of infective endocarditis in catheterized ACE KO, WT, and NeuACE mice as indicated by Gram staining (n = 10 per group). (E) A representative image of Gram staining shows bacterial accumulation in the heart of a catheterized ACE KO mouse challenged with intravenous MRSA. The blue arrow shows vegetative bacterial growth. Scale bar, 100 μm. (F) Survival of ACE KO, WT, and NeuACE mice after aortic valve damage and MRSA challenge is shown (n = 5 per group). A one-way (B) or two-way ANOVA (C) with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Staining, Bacteria

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) Phospho-PKC isoforms were measured by ELISA in ACE KO, WT, and NeuACE bone marrow neutrophils after stimulation with LPS for 30 min in vitro (n = 8 per group). (B) Phospho–p38-MAPK in bone marrow neutrophils was measured by ELISA after stimulation with LPS for 30 min in vitro (n = 8 per group). Mice were treated with ramipril or losartan for 1 week before neutrophil isolation. (C) Western blotting of ACE KO, WT, and NeuACE neutrophils for phospho–p38-MAPK is shown. β-Actin serves as the loading control. Numbers below bands indicate relative band density as determined by Image Studio Lite version 5.2 (LI-COR). (D) Superoxide (O −2) concentration in neutrophils after stimulation with LPS (1 μg/ml) for 10 min at room temperature was measured using a cytochrome C reduction assay (n = 8 per group). For p38-MAPK inhibition, neutrophils were pretreated with 500 nM SB203580 for 2 hours at room temperature. (E) Measurement of phospho–p47-phox in neutrophils treated with or without SB203580 and stimulated with LPS by Western blot. TAP2 serves as the loading control. Numbers below bands indicate relative band density. (F) Ex vivo MRSA clearance by blood from ACE KO, WT, and NeuACE mice after treatment with SB203580 is shown. Blood was drawn from mice and mixed with SB203580 for 1 hour. Then, after addition of MRSA (1.4 × 106 CFU/ml), blood samples were assessed for their ability to eliminate MRSA after a 2-hour incubation (n = 5). (G) In vitro intracellular killing of MRSA in neutrophils purified from bone marrow (n = 6 per group). One-way (A) or two-way ANOVA (B, D, F, and G) with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Isolation, Western Blot, Control, Concentration Assay, Inhibition, Ex Vivo, Incubation, Purification

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: (A) Bone marrow neutrophils were cultured and stimulated with LPS (500 ng/ml) for 12 hours, and then, the supernatant plus cells were collected for ELISA. Groups of mice were treated with either ramipril (40 mg/liter) or losartan (600 mg/liter) in drinking water for 1 week before isolation of bone marrow neutrophils, and then, cells were treated with 10 μM ramipril or 100 μM losartan during the in vitro assay. (B) Measurement of LTB4 in serum isolated 24 hours after challenge with intravenous MRSA (1.2 × 108 CFU/100 μl). (C) Measurement of ROS production in neutrophils treated with LPS (1 μg/ml) for 30 min using 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) staining and flow cytometry. A representative flow cytometry plot is shown on the left, and quantification of mean fluorescence intensity (MFI) of ROS indicator 2’,7’-dichlorodihydrofluorescein (DCF) is shown on the right. To inhibit LTB4 synthesis, groups of mice were treated with the 5-LOX inhibitor zileuton (5 mg/kg per day, gavage) for 2 days before isolation of bone marrow neutrophils, and the drug was maintained at a concentration of 100 μM during in vitro assays. (D) Mice were pretreated with zileuton, and bone marrow–isolated neutrophils were challenged in vitro with MRSA at MOI 13 to evaluate intracellular killing. (E) Neutrophils were preincubated with 100 μM zileuton and then placed in the top chamber of a transwell to measure chemotaxis. The cells that migrated to the bottom chamber in response to 100 nM fMLP were collected and enumerated by cell counter. (F) Bone marrow neutrophils were cultured and stimulated with 100 nM fMLP for 12 hours. Supernatant along with cells were collected for ELISA to measure MIP-2 and IL-1β concentrations. Two-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons, and data are presented as means ± SEM (n = 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, In Vitro, Staining, Flow Cytometry, Fluorescence, Concentration Assay, Chemotaxis Assay

Journal: Science translational medicine

Article Title: An ACE inhibitor reduces bactericidal activity of human neutrophils in vitro and impairs mouse neutrophil activity in vivo

doi: 10.1126/scitranslmed.abj2138

Figure Lengend Snippet: Blood was drawn from study participants before ramipril (Base), after ramipril administration (Ram), and after a 1-week washout (Wash). (A) After in vitro infection of blood with MRSA (about 1 × 106 CFU/ml), K. pneumoniae (about 1 × 106 CFU/ml), or P. aeruginosa (about 1 × 106 CFU/ml), samples were assessed for their ability to eliminate bacteria after 2- or 5-hour incubations relative to 0 hours. (B) In vitro intracellular killing of bacteria by purified neutrophils at 2 and 5 hours after infection with either MRSA, K. pneumoniae, or P. aeruginosa at an MOI of about 15 is shown. (C) Neutrophils were isolated from participants and stimulated with LPS (1 μg/ml), and ROS production was measured using the DCFDA Cellular ROS Assay Kit. The MFI of the ROS indicator, 2’,7’-dichlorodihydrofluorescein (DCF), is shown. (D) Superoxide was measured in neutrophils isolated from participants and stimulated with LPS (1 μg/ml) by cytochrome C reduction assay. Filled box represents the 10-day sample from participant no. 1. Mixed model linear regression, compound symmetry correlation matrix, and Bonferroni correction were used for statistical analyses. Data are presented as means ± SEM (n = 7 participants). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Inhibitor and blocker treatment Mice were treated either with the ACEI ramipril (40 mg/liter; Roxane Laboratories) or lisinopril (40 mg/liter; LKT Laboratories) or the ARB losartan (600 mg/liter; LKT Laboratories) in drinking water for 7 days before MRSA infection in vivo ( 6 , 33 ).

Techniques: In Vitro, Infection, Bacteria, Purification, Isolation, ROS Assay

Journal: Frontiers in Nutrition

Article Title: A novel angiotensin-I-converting enzyme inhibitory peptide from oyster: Simulated gastro-intestinal digestion, molecular docking, inhibition kinetics and antihypertensive effects in rats

doi: 10.3389/fnut.2022.981163

Figure Lengend Snippet: The -CDOCKER_ENERGY scores and lengths of the potential ACEI peptides analyzed by molecular docking.

Article Snippet: The potential ACEI peptides predicted by molecular docking analysis were ultimately synthesized at Chinapeptides Limited Corporation (Shanghai, China) via a solid-phase synthesis method.

Techniques:

Journal: Frontiers in Nutrition

Article Title: A novel angiotensin-I-converting enzyme inhibitory peptide from oyster: Simulated gastro-intestinal digestion, molecular docking, inhibition kinetics and antihypertensive effects in rats

doi: 10.3389/fnut.2022.981163

Figure Lengend Snippet: The ACE inhibition rates of seven potential ACEI peptides at different concentrations (1 and 2 mg/mL).

Article Snippet: The potential ACEI peptides predicted by molecular docking analysis were ultimately synthesized at Chinapeptides Limited Corporation (Shanghai, China) via a solid-phase synthesis method.

Techniques: Inhibition