Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of DCs in vCP172-infected cultures. (A) Immature human DCs were infected with vCP172 (MOI of 10). Infected (+) and uninfected (−) DCs were cultured for 4 days. After culture, the cells were harvested and monitored for the expression of CD25-, CD83-, and CD86-PE (log PE y axes) versus HLA-DR–FITC (x axes). The percentage of large cells expressing CD25 and CD83 above the isotype control are indicated. (B) Immature rhesus macaque DCs were infected with vCP172 (+) (MOI of 10) or not (−) and cultured for 1 to 3 days before being harvested and analyzed. FACS analysis was performed on cells stained with FITC–anti-HLA-DR versus PE-immunoglobulin, -anti-CD25, -CD80, -CD83, or -CD86. Similar data were obtained from more than five experiments with human DCs and three different monkey donors.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Cell Culture, Expressing, Staining

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Induction of maturation is stimulated by viable canarypox virus. (A) Immature human DCs were infected with an MOI of 10 of either vCP180 or the parental strain (ALVAC) or left uninfected (medium). CD25 surface expression by large HLA-DR-positive cells was assessed 4 days after infection (CD25-PE, y axes; HLA-DR–FITC, x axes). The percentage of CD25-positive cells (above isotype control) are indicated in each panel (highlighted by arrowheads). (B) Immature human DCs that had been infected with vCP180 3 to 4 days earlier were sorted into CD25-negative (CD25 neg.) and CD25-positive (CD25 pos.) fractions. Each fraction was then immunostained for intracellular expression of p27 and analyzed by FACS. The percentage of SIV p27-positive cells, above the immunoglobulin control, is shown for each subset. (C) Immature DCs (human) were infected with live (ALVAC) or heat-inactivated (H.I.) ALVAC or left untreated (medium). After 4 days the DCs were examined for CD25 expression by FACS. The percentages of CD25-positive large cells (compared to the isotype control) are shown in each panel. The CD25-positive subset is highlighted by an arrowhead.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Inhibition of maturation of ALVAC-infected DCs by the addition of a caspase 3 inhibitor. A total of 250 or 500 μM Z-DEVD-FMK, or the equivalent dilution of DMSO diluent for the high dose (DMSO), were added to immature DCs 30 min before infection with ALVAC. Inhibition of maturation was assessed by the lack of CD25 expression 4 days after infection using the DMSO-treated cells as the 100% matured population. The data represent the mean percentages of CD25-expression (% CD25 pos.) of three experiments.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Inhibition, Infection, Expressing

Journal:

Article Title: Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

doi:

Figure Lengend Snippet: Maturation of uninfected DCs by ALVAC-infected immature DCs. Immature DCs were infected with ALVAC, and free virus was washed out. Uninfected immature DCs that had been stained with the green fluorescent dye CMFDA were added to the infected (unstained) DCs at a ratio of 1:1 (Inf. DCs). As controls, the supernatant from infected cells (collected directly after washing off the virus) was added to CMFDA-stained cells (Sup't) or the green-uninfected cells were kept in medium (Medium). Maturation (highlighted by arrowheads) was assessed by CD25 expression 4 days later. The log PE is expressed on the y axes (CD25 versus the IgG control), and the CMFDA fluorescence intensity is expressed on the x axes. The results from one of two similar experiments are provided.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated monoclonal Abs (MAbs) against human major histocompatibility complex (MHC) class II (anti-HLA-DR-FITC) (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, Calif.) were used in combination with phycoerythrin (PE)-conjugated MAbs against human CD25 (BDIS), CD80 (BDIS), CD83 (Coulter Corp., Miami, Fla.), and CD86 (PharMingen, San Diego, Calif.).

Techniques: Infection, Staining, Expressing, Fluorescence

Journal: Journal of Inflammation Research

Article Title: Cerium Oxide-Loaded Exosomes Derived From Regulatory T Cells Ameliorate Inflammatory Bowel Disease by Scavenging Reactive Oxygen Species and Modulating the Inflammatory Response

doi: 10.2147/JIR.S502388

Figure Lengend Snippet: Synthesis and characterization of Treg-exo@nCeO. ( A ) TEM images of nCeO, scale bars: 10 nm. ( B ) EDS spectrum of nCeO. ( C ) Appearance images of nCeO. ( D – F ) hydrogen peroxide and free radical scavenging assay, n=3; *p < 0.05 vs 0 μg/mL, **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( G ) Flow chart of the isolation of CD4+CD25+ T cells. ( H ) Relative proportion of living cells, n=3; **p < 0.01 vs 0 μg/mL, ns, no significance vs 0 μg/mL. ( I ) Uptaken Cy5-labeled nCeO and fluorescence imaging, scale bars: 2 μm. ( J ) NTA of exo and exo@nCeO. ( K ) Western blot assay of the expression of Calnexin, Tsg101 and CD63 in Tregs and exo. ( L ) TEM images and average size of exo and exo@nCeO, n=3, scale bars: 100 nm. ( M ) Uptaken Dio-exo@Cy5-nCeO and fluorescence imaging, scale bars: 5 μm.

Article Snippet: 1×10 6 Tregs were collected and stained with phycoerythrin (PE) monoclonal antibodies against mouse CD25 (E-AB-F1102D, Elabscience, China) and fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies against mouse CD4 (E-AB-F1097C, Elabscience, China) for 30 min, followed by data collection on a flow cytometer (Beckman Coulter, USA).

Techniques: Isolation, Labeling, Fluorescence, Imaging, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Regulatory T Cells as a Novel Candidate for Cell-Based Therapy in Kidney Disease

doi: 10.3389/fphys.2020.00621

Figure Lengend Snippet: (A) Histology of kidney with Adriamycin-induced nephropathy showed severe structural injury, in contrast to healthy control kidney. (B–D) Serum creatinine, urine protein, and creatinine clearance were assessed in control mice and mice with Adriamycin-induced nephropathy. (E) Dot plots from flow cytometry showing numbers of CD4+ CD25+ Tregs isolated from control kidney and kidney with Adriamycin-induced nephropathy. Percentages of cells are indicated within each quadrant. LL, lower left; LR, lower right; Q, quadrant; UL, upper left; UR, upper right.

Article Snippet: The surface of CD3+ cells was labeled with antibodies against CD25 and FOXP3 (BD Accuri C6, United States).

Techniques: Flow Cytometry, Isolation

Journal: Frontiers in Physiology

Article Title: Regulatory T Cells as a Novel Candidate for Cell-Based Therapy in Kidney Disease

doi: 10.3389/fphys.2020.00621

Figure Lengend Snippet: (A) Dot plots from flow cytometry showing numbers of CD4+ CD25+ Tregs isolated from spleens of healthy mice. (B) Increase in Treg numbers after in vitro amplification. (C) Dot plots from flow cytometry showing purity of CD4+ CD25+ Tregs after amplification. (D) Flow cytometry histogram depicting Treg numbers isolated from mice with Adriamycin-induced nephropathy in which Tregs from healthy donors had been adoptively transferred. (E) Fluorescence images of kidneys from control mice, mice with Adriamycin-induced nephropathy, and mice with nephropathy after treatment with Tregs from healthy donors. (F) Histological examination suggested that kidney with AN treated with Tregs show less damage than AN. AN, Adriamycin-induced nephropathy.

Article Snippet: The surface of CD3+ cells was labeled with antibodies against CD25 and FOXP3 (BD Accuri C6, United States).

Techniques: Flow Cytometry, Isolation, In Vitro, Amplification, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleinopathies

doi: 10.1523/JNEUROSCI.1170-17.2017

Figure Lengend Snippet: Effects of combined GP+RAP/α-syn vaccination on the trafficking of T and B cells in the brains of α-syn tg mice. Brain sections from α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP were analyzed immunohistochemically for markers of T cells (CD4), Tregs (CD25, FOXP3), and B cells (CD20). A–F, There was a significant increase in the numbers of the CD4-immunoreactive T cells (A, B), the CD25 cells (C, D), and the FOXP3 markers of Tregs (E, F) in α-syn tg mice treated with either GP+RAP or GP+RAP/α-syn compared with non-tg mice treated with GP-alone. G, H, There was no change in the CD20 marker for B cells across any of the treatment groups. p < 0.05 using ANOVA followed by Dunnett's post hoc test comparing GP+RAP/α-syn (&) or GP+RAP (#) treatment with all other groups. N = 6 mice/group. Scale bar, 25 μm.

Article Snippet: To evaluate the effects of the combined vaccine of immunological markers, the blots were probed with antibodies against CD25 (1:1000; monoclonal, MBL); TGF-β1 (polyclonal, 1:2500, Abcam); CD68 (1:1000; monoclonal, Abcam), TNF-α (1:2000; polyclonal, Abcam), and IL-6 (1:1000; polyclonal, Santa Cruz Biotechnology).

Techniques: Marker

Journal: The Journal of Neuroscience

Article Title: Combined Active Humoral and Cellular Immunization Approaches for the Treatment of Synucleinopathies

doi: 10.1523/JNEUROSCI.1170-17.2017

Figure Lengend Snippet: Verification of the effects of combined GP+RAP/α-syn vaccination on α-synuclein and immune activation markers by immunoblot. Brain homogenates from α-syn tg mice immunized with GP, GP-α-syn, GP+RAP, or GP+RAP/α-syn and non-tg control mice immunized with GP were fractioned and analyzed by Western blot. A–G, Representative Western blot images (A) and quantitative analysis for CD25 (B), FOXP3 (C), α-syn (D), TNF-α (E), IL-6 (F), and TGF-β1 (G) normalized to actin. N = 6 mice/group. p < 0.05 using ANOVA followed by Dunnett's post hoc test comparing each of the other groups with GP-alone treated tg mice (*), GP+RAP/α-syn (&), or GP+RAP (#) treatment.

Article Snippet: To evaluate the effects of the combined vaccine of immunological markers, the blots were probed with antibodies against CD25 (1:1000; monoclonal, MBL); TGF-β1 (polyclonal, 1:2500, Abcam); CD68 (1:1000; monoclonal, Abcam), TNF-α (1:2000; polyclonal, Abcam), and IL-6 (1:1000; polyclonal, Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot