Journal: Journal for Immunotherapy of Cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/mL solution of recombinant human CD80 or CD86 (9050-B1-100 or 9090-B2-100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicles Coordinate Bacterial Cloaking in Lung Epithelial Cells to Alleviate Acute Inflammatory Injury

doi: 10.1002/jev2.70238

Figure Lengend Snippet: The internalization of AEVs‐S. aureus complexes by host cells through the RhoA‐ROCK1‐actin‐driven endocytosis pathway. (A) Representative fluorescence images and quantitative analysis of intracellular S. aureus (red) level in A549 cells across treatment groups ( n = 6). Scale bar, 25 µm. (B) Quantification of intracellular S. aureus level in A549 cells across defined treatment groups post‐infection ( n = 6). (C) Quantification of S. aureus level in A549 cells with or without bafilomycin A1 treatment across SA and AEVs‐SA groups ( n = 6). (D) Western blot analysis of phospho‐CAV‐1, CAV‐1 and ROCK1 in A549 cell lysates collected at 3 h.p.i. across experimental groups. (E) Western blot analysis of phospho‐FAK, phospho‐Src, FAK and Src in A549 cell lysates at 3 h.p.i. ( n = 3). (F) Quantification of intracellular S. aureus level in SA and AEVs‐SA groups after cell surface HSPG antagonism via heparan sulfate ( n = 6). (G) Quantification of intracellular S. aureus level in SA and AEVs‐SA groups after HSPG enzymatic removal by Heparinase III ( n = 6). (H) Quantification of intracellular S. aureus level in AEVs‐SA groups after cell surface cholesterol removal with mβCD and lipid removal with heparin ( n = 6). (I) Comparison of intracellular bacterial load in A549 cells treated with AEVs‐SA following pharmacological inhibition of RhoA, ROCK1, or actin polymerization ( n = 6). (J) Representative fluorescence images and quantitative analysis of intracellular S. aureus (red) level in Y‐27632‐treated and untreated A549 cells ( n = 6). Scale bar, 25 µm. (K) Western blot analysis of ROCK1 in control and knockout A549 cells. (L) Quantification of intracellular S. aureus level in EVs‐SA group before and after ROCK1 knockout ( n = 6). (M) Quantification of lung tissue‐resident bacteria at 24 h.p.i., following ROCK1 inhibition ( n = 6). (N) Survival analysis of lethally infected mice (6 × 10 8 CFU S. aureus ) in different groups ( n = 10). SA denotes Staphylococcus aureus . AEVs‐SA indicates AEVs‐S. aureus complexes. Data are presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA with Tukey's post hoc test (A–M) and log‐rank test (N). * P < 0.05, ** P < 0.01.

Article Snippet: Pharmacological treatments included: Bafilomycin A1 (MedChemExpress, HY‐100558; 10 nM, 30 min), GW4869 (MedChemExpress, HY‐19363; 20 nM, 10 h), Y‐27632 (MedChemExpress, HY‐10071; 1 μM, 30 min), Heparan sulfate (MedChemExpress, HY‐101916; 100 μg/mL, 30 min), Heparin (MedChemExpress, HY‐17567A; 10 μg/mL, 4 h), Cytochalasin D (MedChemExpress, HY‐N6682; 1 μg/mL, 4 h), Chlorpromazine (MedChemExpress, HY‐12708; 10 μg/mL, 30 min), Nystatin (MedChemExpress, HY‐17409; 25 μg/mL, 30 min), Amiloride (MedChemExpress, HY‐B0285; 1 mmol/L, 30 min), Heparinase III (New England Biolabs, P0737S; 10 mU, 30 min), Rho Inhibitor I (Cytoskeleton, CT04; 0.5 μg/mL, 4 h), SPA (Sino Biological, 10600‐P07E; 100 μg/mL, 12 h).

Techniques: Fluorescence, Infection, Western Blot, Comparison, Inhibition, Control, Knock-Out, Bacteria

Journal: bioRxiv

Article Title: Functional cardiac consequences of β-adrenergic stress-induced injury in the mdx mouse model of Duchenne muscular dystrophy

doi: 10.1101/2024.04.15.589650

Figure Lengend Snippet: Isoproterenol promotes sarcolemmal injury in dystrophin-deficient cardiac myocytes. The acute and chronic effects of isoproterenol (2 mg/kg/day) were assessed in mdx and wild-type (WT) mice treated for 1- (n=10 per group) or 10 days (n=5 per group). Control mice were injected with an equal volume of saline for 10 days (n=5 mdx and n=4 WT). (A-D) To assess sarcolemmal damage, mid-chamber coronal sections of ventricles were immunolabeled with anti-IgM (red) and counterstained with wheat germ agglutinin (WGA; green) to visualize tissue morphology. (A) Cardiac injury was detected transiently in mdx mice after isoproterenol challenge with IgM+ cardiac myocytes occupying ∼8.5% of the myocardial area after a single isoproterenol treatment. Cardiac myocyte injury was scarcely detected in mdx mice after chronic isoproterenol administration or in WT mice across all conditions. (B-C) Representative mdx (B) and WT (C) whole ventricle cross-section and high magnification images show prominent areas of injury (red) after acute isoproterenol treatment in mdx mice. Bar=500 µm. (C) High-magnification images confirm that the IgM+ signal is localized to cardiac myocytes in mdx mice. (D) Serum cardiac troponin I (cTnI) levels were measured by ELISA as an independent assay of cardiac injury. Serum cTnI levels were also transiently elevated in mdx mice after acute isoproterenol treatment (n=5 per group). Data are presented as mean ± SEM. All p values are based on ANOVA with Tukey’s multiple comparison test. ** p <0.01 and *** p <0.001 versus WT within a treatment condition. ## indicates p <0.01 between treatment groups within a genotype.

Article Snippet: For the imaging portion of the study, female wild-type (n=10) and mdx mice (n=10), 10-12 weeks of age, similarly received daily subcutaneous injections of 2 mg/kg/day isoproterenol (MedChemExpress #HY-B0468).

Techniques: Control, Injection, Saline, Immunolabeling, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: Functional cardiac consequences of β-adrenergic stress-induced injury in the mdx mouse model of Duchenne muscular dystrophy

doi: 10.1101/2024.04.15.589650

Figure Lengend Snippet: Distinct cardiac growth and stress responses to β-adrenergic stimulation in dystrophic hearts. Heart weight normalized to tibia length (HW/TL) and heart weight normalized to body mass (HW/BM) were used as indices of cardiac growth in mdx and WT mice treated for 1- (n=10 per group) or 10-days (n=5 per group), and control mice (n=5 mdx and n=4 WT). (A) HW/TL ratio was higher in mdx mice compared to WT in all conditions. Chronic isoproterenol treatment resulted in an increased HW/TL ratio in WT mice only. (B) HW/BM ratio increased with chronic isoproterenol treatment in WT mice relative to control and mdx mice. (C-D) QPCR analyses of mdx and WT ventricles expressed as mRNA levels relative to WT control. (C) Nppa and (B) Nppb mRNA expression increased in mdx mice with chronic isoproterenol challenge and relative to WT. (E) Ventricular wall thickness was measured at key time points by ultrasound (n=10 per genotype). At baseline, wall thickness was greater in mdx hearts relative to WT. However, in response to isoproterenol treatment wall thickness decreased in mdx mice becoming less thick than WT at day 14. Mid-chamber coronal sections of mdx (F) and WT (G) ventricles stained with Masson’s trichrome show characteristic fibrosis and thinning of the free wall in mdx mice at day 14. Bar=1 mm. Data are presented as mean±SEM. All p values are based on 2-way ANOVA with Tukey’s multiple comparison test. * p <0.05, ** p <0.01, *** p <0.001 and *** p <0.0001 versus WT within a treatment condition. # p <0.05, ## p <0.01, ### p <0.001 and #### p <0.0001 between treatment groups within a genotype.

Article Snippet: For the imaging portion of the study, female wild-type (n=10) and mdx mice (n=10), 10-12 weeks of age, similarly received daily subcutaneous injections of 2 mg/kg/day isoproterenol (MedChemExpress #HY-B0468).

Techniques: Control, Expressing, Staining, Comparison

Journal: Journal of visualized experiments : JoVE

Article Title: A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

doi: 10.3791/62694

Figure Lengend Snippet: Materials

Article Snippet: Tiletamine + Zolazepam , Zoetis Inc , 54771-9050-1 , Medication, Pre-Operative.

Techniques: Saline, Adhesive, Sterility, Ointment, Control, Blocking Assay