Journal: Animals : an open access journal from MDPI

Article Title: New Insights into the Effects of Microbial Muramidase Addition in the Diets of Broiler Chickens.

doi: 10.3390/ani13081356

Figure Lengend Snippet: Figure 3. The morphometric analytic data of immunostained CD3 and CD20 cells in the spleen of different experimental groups. Means with different superscripts (a, b, c, d) are significantly different at p < 0.05.

Article Snippet: Slides were treated with mouse anti-Chicken CD3, clone CT-3 (Bio-Rad Lab., Dubai, United Arab Emirates), and CD20 (ThermoFisher Scientific, Waltham, MA, USA) and examined as described by Amer et al. [36].

Techniques:

Journal: Animals : an open access journal from MDPI

Article Title: New Insights into the Effects of Microbial Muramidase Addition in the Diets of Broiler Chickens.

doi: 10.3390/ani13081356

Figure Lengend Snippet: Figure 4. The immunostained positive CD3 cells (red arrows) and negative cells (black arrows) in the spleen of different experimental groups. (A): MUR0, (B): MUR200, (C) MUR400 and (D): MUR600.

Article Snippet: Slides were treated with mouse anti-Chicken CD3, clone CT-3 (Bio-Rad Lab., Dubai, United Arab Emirates), and CD20 (ThermoFisher Scientific, Waltham, MA, USA) and examined as described by Amer et al. [36].

Techniques:

Journal: Journal of Veterinary Science

Article Title: Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

doi: 10.4142/jvs.2013.14.1.53

Figure Lengend Snippet: Percentages of CD3+CD4+ and CD3+CD8+ T lymphocytes in the different groups of chickens. Columns are labeled with letters. Different letters indicate statistically significant differences ( p < 0.01) while letters in common indicated that no significant differences ( p > 0.01) were observed in each T cell subgroup.

Article Snippet: The samples (100 µL; 1 × 10 6 cells) was incubated with mouse anti-chicken CD4-PE (BD Biosciences, USA), mouse anti-chicken CD3-SPRD (BD Biosciences, USA), and mouse anti-chicken CD8-FITC (BD Biosciences, USA) primary antibodies (antibody final concentrations as 1.25 µg/100 µL) in the dark at 4℃ for 30 min. After the incubation, all samples were washed three times with cold PBS and then resuspended in 0.5 mL of PBS.

Techniques: Labeling

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infection upregulates COX-2 expression and impairs T cell proliferation. (A) Graphical presentation for experimental setup for ex vivo proliferation assay: Splenocytes, harvested at 21 dpi from control, MDV infected (RB1B, 1,000 pfu/dose), and vaccinated birds (Rispens-CVI988, 1,000 pfu/dose), were stained with CFSE and stimulated with Con-A (5 µg/ml) for 3 days. (B) Representative dot plot and histograms showing the gating strategy used in acquisition and analysis of CFSE based proliferation data. The proliferation of 7AAD - (live cells) CD3 + T cells was analysed by flow cytometry. (C) Proliferation index showing proliferation in CD3+ T cells from control, MDV infected and vaccinated birds. (D) qRT-PCR data showing fold change in mRNA level for COX-2 gene in MDV infected and vaccinated birds which was calculated over the control birds. (E) Representative histograms and corresponding (F) proliferation index showing proliferation in CD3+ T cells from MDV infected birds stimulated with Con-A in presence of the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). Grey bars: No significant difference between mock and treatment and red bars: significant difference or restoration to non-significant level between mock and treatment group. Dot plot and histograms are representative of proliferation data from six individual infected birds. Each dot in proliferation index represents the average of three biological replicates from six individual chickens. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Expressing, Ex Vivo, Proliferation Assay, Control, Staining, Flow Cytometry, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infected CEFs and 265L tumour cells produce high PGE2 level resulting in downregulation of chIL2 and transferrin uptake in CD3+ T cells. Culture supernatants were harvested from control CEF (non-infected),CEF infected with virulent MDV (MDV supernatant) or vaccine strain of MDV (vaccine supernatant) at 72 hrs post-infection. Culture supernatant harvested from CD4+ T cells of naïve splenocytes and MDV-transformed CD4+ T cells (265L supernatant). (A) The levels of PGE2 were determined using an ELISA assay. (B) chIL2 expression level was determined in Con-A stimulated splenocytes treated with PGE2 (10 µg/ml), or the supernatants as described above using qRT-PCR, and fold change are shown. Inhibition of chIL-2 expression by (C) PGE2, (D) MDV supernatant, (E) 265L supernatant was rescued in the presence of the inhibitors of the COX-2/PGE2 pathway; TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). (F) Representative confocal images showing uptake of transferrin (red color) by CD3+ T cells in the Con-A stimulated splenocytes treated with control supernatant, PGE2, MDV supernatant, vaccine supernatant or 265L supernatant using confocal microscopy. (G) Graph shows mean fluorescent intensity (MFI) of transferrin uptake using confocal microscopy. (H) Bar graph shows the effects of a COX-2 inhibitor (SC-236) on transferrin uptake in CD3+ T cells treated with PGE2, MDV supernatant or 265L supernatant using confocal microscopy. Graphs are representative of three independent experiments, each performed with three biological replicates. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot in ELISA represents the average of three biological replicates from six individual culture groups. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Control, Transformation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Inhibition, Confocal Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Expression of CD25 + on CD3 + T cells remained unaffected by PGE2 treatment. CD25 surface expression on CD3+ T cells were quantified in splenocytes stimulated with Con-A (5 µg/ml) in presence of cell culture media containing either recombinant PGE2 (5 µg/ml), the control supernatants, MDV supernatant, vaccine supernatant or the 265L supernatant. (A) Dot plot (B) bar graph showing percentage of CD3 + CD25 + T cells. Graph is representative of three independent experiments, each performed with three biological replicates per treatment. Each dot in ELISA represents the average of three biological replicates from six individual culture groups. Grey bars represent the experimental groups with no significant difference. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Expressing, Cell Culture, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infection drives PGE2/COX-2 dependent downregulation of T cell proliferation in vitro . Splenocytes were harvested from naïve birds and stained with CFSE and then they were stimulated with Con-A (5 µg/ml) in presence of PGE2, control supernatant, MDV supernatant, Vaccine supernatant, or 265L supernatant with or without the chemical the inhibitors of the PGE2-COX-2 pathway. After 3 days of culture, T cell proliferation was analysed by flow cytometry. (A) Representative histograms and corresponding (B) proliferation index showing dose-dependent effect of recombinant PGE2 (2.5, 5 and 10 µg/ml) on in vitro proliferation of CD3+ T cells. (C) Representative histograms and corresponding (D) proliferation index showing effect of control supernatant, MDV supernatant, Vaccine supernatant, or 256L supernatant on in vitro proliferation of CD3+ T cells. (E, G) Representative histograms and corresponding (F, H) proliferation index showing inhibitory effect of the chemical inhibitor: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). Dot plot and histograms are representative of proliferation data from six individual birds. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot in proliferation index represents the average of three biological replicates from six individual chickens. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, In Vitro, Staining, Control, Flow Cytometry, Recombinant, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Treatment of splenocytes with PGE2/MDV sup/265L sup induced PGE2-COX-2 pathway drives dysfunction of CD3 + T cells. (A) Splenocytes were cultured for 3 days in media containing recombinant PGE2 (5 µg/ml), control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The isolated CD3 + T cells were CFSE stained and co-cultured with freshly isolated CD3 negative cells. The co-culture was stimulated with Con-A (5 µg/ml) in presence or absence of the chemical inhibitors of the PGE2-COX-2 pathway. After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B) Representative histograms and corresponding (C) proliferation index. (D) Representative histograms and corresponding (E–G) proliferation index showing in vitro proliferation in CD3 + T cells from splenocytes cultured with PGE2, MDV Supernatant and 265L supernatant in presence of the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL), respectively. Data is representation of three independent experiments, each performed with three biological replicates per treatment. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Statistical significance was estimated by p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Cell Culture, Recombinant, Control, Isolation, Staining, Co-Culture Assay, Flow Cytometry, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: PGE2-COX-2 drives dysfunction of CD3 + T cells in MDV infected chickens. (A) CD3 + T cells were isolated from splenocytes harvested at 21 dpi from control (n=5), MDV infected (n=5) and vaccinated birds (n=5) and stained with CFSE before co-culturing with CD3 negative T cells isolated from splenocytes of naïve birds. The co-culture was stimulated with Con-A (5 µg/ml) in presence or absence of the chemical inhibitors of PGE2-COX-2 pathway. After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B, D) Representative histograms showing proliferation in CD3+ T cells from the control, vaccinated birds, and MDV infected birds with or without the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL) and corresponding (C) proliferation index from the experimental groups of birds. (E) Proliferation index for CD3 + T cells from the MDV infected birds in presence of the chemical inhibitors of the PGE2-COX-2 pathway. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Statistical significance was estimated by p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Isolation, Control, Staining, Co-Culture Assay, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: PGE2/MDV supernatant directly affected CD3+ T cells and impaired CD3 + T cells is independent of CD3 negative T cells and BMDCs. (A) CD3 negative cells were isolated from splenocytes cultured for 3 days in media containing recombinant PGE2 (5 µg/ml), the control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The isolated CD3 negative cells were co-cultured with CFSE stained CD3 + T cells isolated from naive birds in presence of Con-A (5 µg/ml). After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B, C) Representative histograms and corresponding (C) proliferation index showing in vitro proliferation of CD3+ T cells from the experimental groups. (D) BMDCs were cultured for 3 days with media containing recombinant PGE2 (5 µg/ml), the control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The treated BMDCs were harvested, washed, and cultured with CFSE stained CD3 + T cells isolated from naïve birds in fresh medium containing Con-A (5 µg/ml) for 3 days. (E) Representative histograms and corresponding (F) proliferation index for CD3 + T cells from the different experimental groups. Data is representation of three independent experiments, each performed with three biological replicates per treatment. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Statistical significance was estimated by p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Isolation, Cell Culture, Recombinant, Control, Staining, Co-Culture Assay, Flow Cytometry, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Administration of meloxicam rescues T cell proliferation in MDV infected chickens. (A) Schematic diagram showing the experimental groups; non-infected mock controls, MDV infected birds (RB1B, 1,000 pfu/dose), and MDV infected birds that received daily oral administration of meloxicam. (B) COX-2 expression levels in splenocytes of the experimental groups were analysed using RT-PCR assay. CFSE-labelled splenocytes were stimulated with Con-A and cultured for 3 days in vitro to assess T cell proliferation using flow cytometry. (C) Representative histograms and corresponding (D) proliferation index showing ex vivo proliferation of CD3+ T cells in mock control and MDV infected with and without meloxicam treated birds. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Statistical significance was estimated by p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, In Vitro, Flow Cytometry, Ex Vivo, Control, Standard Deviation