Journal: Infection and Immunity

Article Title: Role of T Cells and Gamma Interferon during Induction of Hypersensitivity to Lipopolysaccharide by Toxic Shock Syndrome Toxin 1 in Mice

doi: 10.1128/iai.69.3.1256-1264.2001

Figure Lengend Snippet: FIG. 4. Effects of Cs (A) or anti-IFN-g MAb (B) on LPS-induced serum TNF-a activity in BALB/c-AnNCr mice primed with TSST-1. Groups of four mice were pretreated with Cs (40 mg/kg, i.p.) or anti-IFN-g MAb (750 mg/mouse, i.p.) at 4 or 2 h, respectively, prior to injection of TSST-1. Mice were then injected i.p. with 200 mg of TSST-1 (dotted bars) per kg or PBS (clear bars). Control mice received equal volumes of PBS instead of Cs, rat IgG2a instead of anti-IFN-g MAb, or PBS instead of TSST-1. All mice were injected with LPS (400 mg/kg, i.p.) 12 h later. TNF-a activity in serum was measured 2 h postinjection of LPS. Solutions of Cs or MAb were treated with 2 mg of polymyxin B per ml to remove contaminating LPS. The percent reductions in circulating TNF-a activity caused by Cs or anti-IFN-g MAb are shown.

Article Snippet: Neutralizing rat monoclonal antibody (MAb) to mouse IFN-g, rat immunoglobulin G2a (IgG2a) isotype control MAb, and murine recombinant TNF-a were obtained from R&D Systems (Minneapolis, Minn.).

Techniques: Activity Assay, Injection, Control

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 μg/10 6 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: JCI insight

Article Title: NK cells contribute to reovirus-induced IFN responses and loss of tolerance to dietary antigen.

doi: 10.1172/jci.insight.159823

Figure Lengend Snippet: Figure 5. NK cells are dispensable for viral clearance but contribute to IFN-mediated inflammatory responses that are associated with loss of tolerance to dietary antigen. WT mice were i.p. injected with either isotype control IgG2a Ab or anti-NK1.1 Ab (PK136) 1 day prior to and 1 day following PO inoculation with 1 × 108 PFU of T1L or PBS as a control. At 2 dpi, MLNs were resected and processed for flow cytometry (n = 8–10). (A) Design of NK cell depletion studies. (B) Viral titers in MLNs and a 3 cm section of the ileum determined by plaque assay at 2 dpi. (C) Model of immune pathways involved in T1L-induced LOT made with Biorender. (D) Type I and type II IFN–dependent gene expression compared with GAPDH in MLNs determined by qPCR at 2 dpi. Results are presented as mean values. Data are shown as mean ± SEM. Statistical significance was calculated using Student’s t test in B and 1-way ANOVA with Tukey’s multiple comparisons test in D. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: To deplete IL-12, mice were i.p. injected with 750 μg of anti–IL-12p40 depleting Ab (C17.8, Bio X Cell) or InVivoPlus mouse IgG2a isotype control (Bio X Cell) following previously established protocols (65–67).

Techniques: Injection, Control, Flow Cytometry, Plaque Assay, Gene Expression

Journal: JCI insight

Article Title: NK cells contribute to reovirus-induced IFN responses and loss of tolerance to dietary antigen.

doi: 10.1172/jci.insight.159823

Figure Lengend Snippet: Figure 6. NK cells contribute to CD103+CD11b– DC responses and inflammatory activation in the MLNs. WT mice were i.p. injected with either isotype control IgG2a Ab or anti-NK1.1 Ab (PK136) 1 day prior to and 1 day following PO inoculation with 1 × 108 PFU of T1L or PBS as a control. At 2 dpi, MLNs were resected and processed for flow cytometry. Single-cell suspensions were incubated with brefeldin A in the presence of Golgi Plug at 37°C for 6 hours (n = 6–8). (A) Model of inflammatory DC activation important in LOT to dietary antigen made with Biorender. (B) Total number of CD103+CD11b– DCs that express CD8α or CD86. (C) Percentage and total number of migratory tolerogenic DCs that express intracellular IL-12p40. Results are presented as mean values. Data are shown as mean ± SEM. Statistical significance was calculated using a 1-way ANOVA with Tukey’s multiple comparisons test in B and C. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: To deplete IL-12, mice were i.p. injected with 750 μg of anti–IL-12p40 depleting Ab (C17.8, Bio X Cell) or InVivoPlus mouse IgG2a isotype control (Bio X Cell) following previously established protocols (65–67).

Techniques: Activation Assay, Injection, Control, Flow Cytometry, Incubation

Journal: JCI insight

Article Title: NK cells contribute to reovirus-induced IFN responses and loss of tolerance to dietary antigen.

doi: 10.1172/jci.insight.159823

Figure Lengend Snippet: Figure 7. NK cells contribute to antiviral host CD4 Th1 T cell responses. WT mice were injected i.p. with either isotype control IgG2a Ab or anti-NK1.1 Ab (PK136) 1 day prior to and 1 day following PO inoculation with 1 × 108 PFU of T1L or PBS as a control. OT-II T cells were transferred from CD45.1+ WT mice to CD45.2+ WT mice 1 d prior to reovirus inoculation. At 3 dpi, MLNs were resected and processed for flow cytometry (n = 5–6). (A) Schematic of experimental procedure made with Biorender. (B) Reovirus gene transcripts (S4 gene) in MLNs determined by qPCR at 3 dpi. (C) Total number and frequency of host Th1 (Tbet+, Foxp3–) CD4+ T cells (CD45.1– CD4+). (D) Total number and frequency of Treg (Tbet–, Foxp3+) host CD4+ T cells (CD45.1– CD4+). Results are presented as mean values. Data are shown as mean ± SEM. Statistical significance was calculated using Student’s t test in B and 1-way ANOVA with Tukey’s multiple comparisons test in C and D. *P < 0.05; **P < 0.01.

Article Snippet: To deplete IL-12, mice were i.p. injected with 750 μg of anti–IL-12p40 depleting Ab (C17.8, Bio X Cell) or InVivoPlus mouse IgG2a isotype control (Bio X Cell) following previously established protocols (65–67).

Techniques: Injection, Control, Flow Cytometry

Journal: JCI insight

Article Title: NK cells contribute to reovirus-induced IFN responses and loss of tolerance to dietary antigen.

doi: 10.1172/jci.insight.159823

Figure Lengend Snippet: Figure 8. NK cells contribute to T1L-induced T cell priming responses associated with LOT to dietary antigen early following inoculation. WT mice i.p. injected with either isotype control IgG2a Ab or anti-NK1.1 Ab (PK136) 1 day prior to and 1 day following PO inoculation with 1 × 108 PFU of T1L or PBS as a control. OT-II T cells were transferred from CD45.1+ WT mice to CD45.2+ WT mice 1 day prior to reovirus inoculation. At 3 dpi, MLNs were resected and processed for flow cytometry (n = 5–6). (A) Contour plot of OT-II T cell expression of transcription factors Tbet (y-axis), indicating a Th1 phenotype, and Foxp3 (x-axis), indicating a Treg phenotype. (B) Total number and frequency of Treg (Tbet–, Foxp3+) OT-II CD4+ T cells (CD45.1+ CD4+). (C) Total number and frequency of Th1 (Tbet+, Foxp3–) OT-II CD4+ T cells (CD45.1+ CD4+). Results are presented as mean values. Data are shown as mean ± SEM. Statistical significance was calculated using 1-way ANOVA with Tukey’s multiple comparisons test in B and C. *P < 0.05; ****P < 0.0001.

Article Snippet: To deplete IL-12, mice were i.p. injected with 750 μg of anti–IL-12p40 depleting Ab (C17.8, Bio X Cell) or InVivoPlus mouse IgG2a isotype control (Bio X Cell) following previously established protocols (65–67).

Techniques: Injection, Control, Flow Cytometry, Expressing

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: Anti-IL-9 antibody treatment decreased mast cell infiltration of the CNS. Purified cells (mainly composed of mast cells) were harvested from the CNS on days 0, and 5, 15, and 20 after EAE immunization. CD45 + CD117 + mast cells were detected by flow cytometry. CNS mast cells maintained a high level throughout the disease course in EAE group. However, mast cells were significantly reduced from day 5, and remained low in anti-IL-9 Abs group, compared with IgG group. Data are shown as mean ± SE. * P < 0.01, anti-IL-9 Abs group versus IgG group. CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; SE: Standard error; IL: Interleukin.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Purification, Flow Cytometry

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: IL-9 blockade reduced production of chemokine recruiting mast cells in the CNS. Five days after immunization, mRNA expressions of Pecam1, SCF, Vcam-1, CCL2, and CCL5 in CNS tissue of EAE mice were detected by RT-PCR. After IL-9 neutralization, mRNA expressions of CCL5 and Vcam-1 were significantly decreased in anti-IL-9 Abs group, compared with IgG group. * P < 0.01. Pecam1: Platelet and endothelial cell adhesion molecule 1; SCF: Supercoiling factor; Vcam-1: Vascular cell adhesion molecule 1; CCL2: C-C motif chemokine ligand 2; CCL5: C-C motif chemokine ligand 5; CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; IL: Interleukin; RT-PCR: Reverse transcription-polymerase chain reaction; mRNA: Messenger RNA.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Neutralization

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: In vitro , the effect of anti-IL-9 antibody on splenic mast cells. Splenocytes were harvested from experimental autoimmune encephalomyelitis mice 5 days after MOG immunization. After co-culture with anti-IL-9 antibody or anti-mouse IgG for 7 h, mast cell number was counted by flow cytometry. Splenic mast cells cultured with anti-IL-9 antibody showed significantly lower levels in a dose-dependent manner. This trend was particularly evident with anti-IL-9 antibody concentrations up to 20 μg/ml. * P < 0.05; † P < 0.01. IL: Interleukin; MOG: Myelin oligodendrocyte glycoprotein.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Cell Culture