Journal:

Article Title: Neuroprotective autoimmunity: Naturally occurring CD4 + CD25 + regulatory T cells suppress the ability to withstand injury to the central nervous system

doi: 10.1073/pnas.232565399

Figure Lengend Snippet: Depletion of naturally occurring regulatory CD4+CD25+ T cells in BALB/c mice improves neuronal survival after optic nerve crush injury. (A) BALB/c mice were thymectomized (ThyX) 3 days after birth to deplete their regulatory T cells and were subjected as adults to severe unilateral crush injury inflicted on the intraorbital portion of the optic nerve. Surviving neurons were labeled by the application, 3 days before injury, of the neurotracer dye FluoroGold. Significantly more neurons survived in the thymectomized mice than in control (nonthymectomized) age-matched mice (n = 7–8 in each group; P < 0.001; Student's t test). (B) Neuronal survival after optic nerve crush injury in BALB/c nu/nu mice (devoid of T cells) was worse than in wild-type mice of matched background. Endogenous neuroprotection in the nude mice was restored by injection of 5 × 107 wild-type splenocytes. Injection of splenocytes depleted of regulatory CD4+CD25+ T cells increased neuronal survival in these mice beyond even that seen in the wild type (n = 5–6 in each group; P values between different groups, obtained by two-tailed Student's t test, are indicated by asterisks above the graph bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Mouse anti-rat CD4 antibodies conjugated to phycoerythrin (PE) and mouse anti-rat CD25 antibodies conjugated to fluorescein isothiocyanate were purchased from Serotec.

Techniques: Labeling, Injection, Two Tailed Test

Journal:

Article Title: Neuroprotective autoimmunity: Naturally occurring CD4 + CD25 + regulatory T cells suppress the ability to withstand injury to the central nervous system

doi: 10.1073/pnas.232565399

Figure Lengend Snippet: Naturally occurring regulatory CD4+CD25+ T cells diminish spontaneous neuroprotection. BALB/c mice, endowed with the spontaneous ability to evoke a protective beneficial autoimmune response, were injected with 2 × 106 purified activated regulatory CD4+CD25+ T cells. Control mice were injected with 2 × 106 purified activated effector cells (CD4+CD25−) or with PBS. Injection of regulatory T cells had an adverse effect on neuronal survival (more neurons underwent secondary degeneration). The results shown are of one representative experiment of three independent experiments; n = 5–6 mice in each group (P < 0.01 and 0.05; respectively).

Article Snippet: Mouse anti-rat CD4 antibodies conjugated to phycoerythrin (PE) and mouse anti-rat CD25 antibodies conjugated to fluorescein isothiocyanate were purchased from Serotec.

Techniques: Injection, Purification

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Staining, Cytometry, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: NPJ Parkinson's Disease

Article Title: Delphinidin modulates neuroinflammation and behavioral deficits in a Parkinson’s disease mouse model

doi: 10.1038/s41531-025-01244-0

Figure Lengend Snippet: a Quantification of CD4 + cells in the SN. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test: F(3, 12) = 2.156, P = 0.1464, n-numbers: EV veh = 3, EV del = 4, hαSYN veh = 5, hαSYN del = 4 b Quantification of CD8 + cells in the substantia nigra (SN). Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test: F(3, 12) = 5.524, P = 0.0129, n-numbers: EV veh = 3, EV del = 4, hαSYN veh = 5, hαSYN del = 4 c Quantification of CD4 + cells in the striatum. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test: F(3, 11) = 1.215, P = 0.3501, n-numbers: EV veh = 3, EV del = 4, hαSYN veh = 4, hαSYN del = 4 d Quantification of CD8 + cells in the striatum. Statistical analysis by one-way ANOVA followed by Tukey’s multiple comparisons test: F(3, 11) = 0.2133, P = 0.8851, n-numbers: EV veh = 3, EV del = 4, hαSYN veh = 4, hαSYN del = 4 e Representative IF images of CD8 + CD122 + cells in the SN. Scale bar: 50 µm f Quantification of CD8 + CD122 + cells in the SN – as indicated by TH + cells. Statistical analysis by one-tailed multiple t-tests, only showing significant results, P = 0.0425 (hαSYN veh vs. hαSYN del ), P = 0.0270 (EV veh vs. hαSYN del ), n-numbers: EV veh = 6, EV del = 6, hαSYN veh = 6, hαSYN del = 6; *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. Data are shown as min-to-max and mean (+).

Article Snippet: Subsequently, sections were incubated overnight with rat anti-mouse CD4 (1:1000, Bio-Rad, cat. #MCA1767; RRID:AB_322769), rat anti-mouse CD8 (1:500, Bio-Rad, cat. #MCA609G; RRID:AB_321407), rat anti-mouse CD11b (1:100, Bio-Rad, cat. #MCA711; RRID:AB_321292), or rabbit anti-TH antibodies.

Techniques: One-tailed Test

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: Infection with BCG results in accumulation and not deletion of superantigen-responsive CD4+ or CD8+ T cells. Unfractionated splenocytes (5 × 105 cells/tube) from SEB-exposed mice (7 days postinjection) (A) and PBS-injected controls or day-16-infected mice and PBS-injected controls (B) were stained with FITC-labeled anti-mouse Vβ8 TCR (A and B) and either PE-labeled anti-mouse CD4 or anti-mouse CD8. The percentages of Vβ8+ CD4+ T cells (white bars) or Vβ8+ CD8+ T cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Injection, Staining, Labeling, Flow Cytometry

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infectious dose correlates with increased expression of the T-cell activation markers CD44, IL-2Rα, and IL-2Rβ. Unfractionated splenocytes (5 × 105 cells/tube) from each type of spleen were stained with FITC-labeled anti-mouse CD44, anti-mouse CD25/IL-2Rα, or anti-mouse CD122/IL-2Rβ and PE-labeled anti-mouse CD4 or anti-mouse CD8α. The percentages of CD4+ CD44hi, IL-2Rαhi, or IL-2Rβhi CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. Purified CD4+ T cells from the spleens of BCG HD-infected mice (day 16) were stained with FITC anti-mouse CD44 and were sorted in an EPICS Elite ESP (Beckman Coulter) into CD44low (naïve) and CD44hi (memory/effector) populations. Sorted cells were cocultured at 105 cells/well either with bone marrow-derived dendritic cells (5 × 104 cells/well) in the presence or absence of SEB (10 μg/ml) or with M12 B-lymphoma cells (5 × 104 cells/well) in the presence or absence of BCG-Ag (10 μg/ml). IFN-γ levels were determined as described in Materials and Methods. Mean ± standard deviation of triplicate wells is shown.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Expressing, Activation Assay, Staining, Labeling, Flow Cytometry, Purification, Infection, Derivative Assay, Standard Deviation

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: BCG infection induces potent APCs that stimulate rather than inhibit IFN-γ production to SEB and BCG-Ag by purified CD4+ T cells. Sixteen days after injection of mice with PBS or BCG LD or BCG HD, adherent cells were obtained from bulk splenocyte suspensions by panning as described in Materials and Methods. Adherent cell populations (5 × 105 cells/well) were cocultured with purified CD4+ T cells (105 cells/well) from the same mice in the presence of either SEB (10 μg/ml) (A) or BCG-Ag (10 μg/ml) (B). (C) KLH-specific HDK1 type 1 clone was cocultured at 105 cells/well with different numbers of unfractionated splenocytes from PBS control mice or BCG LD- or HD-infected mice in the presence of KLH (10 μg/ml). IFN-γ levels were determined as described in the Fig. ​Fig.11 legend. Mean ± standard deviation of triplicate wells is shown. (D) BCG-infected spleens have more MHC class II+ and Mac1+ cells. Unfractionated splenocytes (5 × 105 cells/tube) from mice that were infected for 2 weeks with BCG LD- or BCG HD-infected mice or PBS-injected controls were stained with FITC-labeled anti-mouse I-Ad/I-Ed/MHC class II and PE-labeled anti-mouse Mac1/CD11b. The percentages of MHC class II+ (white bars) and Mac1+ (black bars) APCs in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Purification, Injection, Standard Deviation, Staining, Labeling, Flow Cytometry

Journal:

Article Title: Mycobacterium bovis BCG-Infected Mice Are More Susceptible to Staphylococcal Enterotoxin B-Mediated Toxic Shock than Uninfected Mice despite Reduced In Vitro Splenocyte Responses to Superantigens †

doi: 10.1128/IAI.70.8.4148-4157.2002

Figure Lengend Snippet: HD splenocytes have a decreased percentage of T cells and show reduced viability early after culture with BCG-Ag and SEB. (A) Unfractionated splenocytes (5 × 105 cells/tube) from mice that were infected for 2 or 3 weeks with BCG LD or BCG HD or from PBS-injected controls were stained with FITC-labeled anti-mouse CD4 and PE-labeled anti-mouse CD8α. The percentages of CD4+ (white bars) or CD8+ cells (black bars) in the spleen were determined by flow cytometry. Means ± standard errors of the means of three experiments are shown. ∗, P < 0.05; ∗∗, P < 0.01. (B) Unfractionated splenocytes were subsequently stimulated with BCG-Ag (10 μg/ml) or SEB (10 μg/ml) at 5 × 105 cells/well in parallel cultures. At 24 h, cells from triplicate wells were pooled and stained with both FITC-labeled anti-mouse CD4 and anti-mouse CD8α and were counterstained with PI (10 μg/ml). The total percentage of nonviable (PI+) T cells was determined by flow cytometry.

Article Snippet: In some experiments, splenocytes that were cultured for 24 or 72 h with SEB or BCG antigen were stained with FITC-conjugated rat anti-mouse CD4 (YTS 191.1) and rat anti-mouse CD8α (IM7.8.1; both from Cedarlane) and counterstained with propidium iodide (PI) (10 μg/ml; Sigma) to enumerate viable cells.

Techniques: Infection, Injection, Staining, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: Cytokines and chemokines were analyzed in colonic tissue from CD200 tg , WT, CD200R1KO and CD200KO mice with DSS-induced acute colitis. A and B, Real-time RT-PCR was performed using total RNA extracted from colonic tissue (5 samples/group) or LP cells (3 samples/group), with data normalized to the expression of GAPDH and HPRT in the same organ. Expression levels in CD200 tg , CD200R1KO and CD200KO mice (mean ± SD in 5 mice) are shown relative to that in WT at day 0 (designated as 1). * p < 0.05; ** p < 0.01; *** p < 0.001 compared to WT control (day 0). (C) Tissue explants from the four experimental groups were cultured and used for multi-analyte Elisarray. Again, relative expression levels of cytokine/chemokine (mean ± SD in 5 mice) in CD200 tg , CD200R1KO and CD200KO mice are shown compared to that in WT at day 0 (designated as 1), using the manufacturer’s software supplied. * p < 0.05; ** p < 0.01; *** p < 0.001, compared to this WT control (day 0). (D) Inflammatory cytokines measured by commercial ELISA (Biolegend: USA) in 36hr culture supernatants of colon-derived CD11c + , CD11b + , or CD4 + T cells (isolated by MACS columns) from WT, CD200 tg , CD200KO or CD200RKO mice 9 days post initiation of DSS treatment. Cells were obtained from a pool of 4 mice/group, and cultured without further stimulation for 36hrs in complete medium. In some cases, as shown, cells were cultured in the presence of CD200Fc (5mg/ml). Data show mean ( + SD) of triplicate measurements. *, indicates p<0.05 compared with WT control; **indicates p<0.05 compared to equivalent group with no CD200Fc. E, Comparison of weight loss in 4 groups of DSS treated mice receiving anti-CD4mAb (right hand panel) or isotype control Ig (left hand panel)-see also . Data are for 4 mice/group, given 50mg Ig at days 4/7 post initiation of DSS treatment. Comparison of equivalent groups in right/left hand panels (Mann Whitney U-test) revealed no statistically significant differences.

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Quantitative RT-PCR, Expressing, Control, Cell Culture, Software, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Comparison, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: mRNAs for cytokines and chemokines were analyzed in colonic tissue harvested 8 days after initiation of the third cycle of DSS-induced chronic colitis from CD200 tg , WT, CD200R1KO and CD200KO mice. Real-time RT-PCR was performed using total RNA extracted from colonic tissue (panel A: 5 samples/group) or LP cells (panel B: 3 samples/group), with data normalized to the expression of GAPDH and HPRT in the same organ. Expression levels in CD200 tg , CD200R1KO and CD200KO mice (mean ± SD in 5 mice) are shown relative to that in WT at day 0 (designated as 1). * p < 0.05; ** p < 0.01; *** p < 0.001, compared with WT control (day 0). (C) Tissue explants from the four experimental groups were cultured and used for multi-analyte Elisarray. Again, relative expression levels of cytokine/chemokine (mean ± SD in 5 mice) in CD200 tg , CD200R1KO and CD200KO mice are shown compared to that in WT at day 0 (designated as 1), using manufacturer’s software. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to WT control (day 0). (D) Inflammatory cytokines measured in 36hr culture supernatants of colon-derived CD11c + , CD11b + , or CD4 + T cells (isolated by MACS columns) from WT, CD200 tg , CD200KO or CD200RKO mice 8 days post initiation of the third cycle of DSS treatment. Cells were obtained from a pool of 4 mice/group, and cultured without further stimulation for 36hrs in complete medium. Data show mean ( + SD) of triplicate measurements. *, indicates p<0.05 compared with WT control-see also .

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Quantitative RT-PCR, Expressing, Control, Cell Culture, Software, Derivative Assay, Isolation

Journal: PLoS ONE

Article Title: Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

doi: 10.1371/journal.pone.0146681

Figure Lengend Snippet: Foxp3, CCR4, CCL-17 and CCL-22 mRNA expression was analyzed in (A) colons and (B) LP cells by real-time RT-PCR from CD200 tg , WT, CD200R1KO and CD200KO mice with DSS-induced chronic colitis. All values were normalized to the expression of GAPDH and HPRT in the same organ with expression levels in CD200 tg , CD200R1KO and CD200KO mice expressed relative to that of WT (designated as 1). Data show mean ± SD for 5 mice/group. * p < 0.05; ** p < 0.01. (C) FACS staining for CD3 + and CD4 + Foxp3 + (gated on CD3 + cells) in mesenteric lymph node (MLN) cells of mice in panels A and B. Data show typical staining patterns from pooled samples (3 mice/group). (D) Loss of protection from weight loss in DSS-induced chronic colitis in CD200 tg mice by treatment with anti-CD25mAb (Fig 7Da), and protection from weight loss in CD200RKO mice by treatment with anti-CD4 mAb (Fig 7Db). Data are shown for 4 mice/group (SD not shown to retain clarity in panels). Mice received 5mg antibody iv 1 day following completion of each DSS treatment. *p<0.05 (Mann-Whitney U-test) compared with equivalent groups not receiving mAb.

Article Snippet: Where CD4 + T cells were depleted from mice with acute DSS-induced colitis, mice received 50mg/mouse of anti-mouse CD4 mAb (GK1.5), purchased from Cedarlane Labs, Hornby, Ontario, iv at 4 and 7 days post DSS initiation, and were sacrificed at 9 days post DSS initiation.

Techniques: Expressing, Quantitative RT-PCR, Staining, MANN-WHITNEY