Journal: Human reproduction (Oxford, England)

Article Title: A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.

doi: 10.1093/humrep/13.4.936

Figure Lengend Snippet: Figure 1. HCL-2 antigen expression in corpus luteum on day 7 detected by indirect immunofluorescence staining. (A) Haematoxylin and eosin staining. (B) Staining with HCL-2 mAb. (C) Staining with anti-apolipoprotein-B mAb (MAB012). (D) Negative control stained with anti-TNP mAb. HCL-2 antigen and apolipoprotein-B were expressed on the granular-shaped structure near the nuclei in the cytoplasm of both large (LL) and small (SL) luteal cells. Both proteins were also detected along the cell membrane, showing the similar expression profiles. Original magnification 3120. Bar 5 100 µm.

Article Snippet: The immunoglobulin isotype was determined using an isotyping kit for mouse monoclonal antibodies (Serotec Ltd, Oxford, UK).

Techniques: Expressing, Staining, Negative Control, Membrane

Journal: Human reproduction (Oxford, England)

Article Title: A monoclonal antibody, HCL-2, raised against human luteal cells reacts with apolipoprotein-B and detects the uptake of low density lipoprotein by luteinizing granulosa cells.

doi: 10.1093/humrep/13.4.936

Figure Lengend Snippet: Figure 5. Detection of HCL-2 antigen on human luteinizing granulosa cells cultured in a medium with or without low density lipoprotein (LDL) by indirect immunofluorescence staining. (A–D) Culture with LDL. (E–H) Culture without LDL. (A, C, E and G) Phase-contrast pictures. (B and F) HCL-2 antigen. (D and H) Negative controls (anti-TNP mAb). HCL-2 antigen was clearly detected in the cytoplasm of human granulosa cells (arrows) cultured in the medium containing LDL for 3 days (B), whereas it was hardly detected in those cultured without LDL (F). Original magnification 3240. Bar 5 50 µm.

Article Snippet: The immunoglobulin isotype was determined using an isotyping kit for mouse monoclonal antibodies (Serotec Ltd, Oxford, UK).

Techniques: Cell Culture, Staining

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: PLoS ONE

Article Title: Regulatory CD4 + Foxp3 + T Cells Control the Severity of Anaphylaxis

doi: 10.1371/journal.pone.0069183

Figure Lengend Snippet: DNP-specific PSA was induced A) in MHC class II-deficient mice (Aβ °/° ) ( triangles ) and WT B6 ( squares ) mice and B) in B6 mice injected with either an anti-CD25 mAb ( triangles ), an anti-CD4 mAb ( circles ) or a control rat IgG mAb ( squares ). All mice were passively sensitized with anti-DNP IgE and challenge with DNP-HSA ( black symbols ) or PBS ( open symbols ). Data show mean (± SEM) values of body temperature at various times after challenge. Figure 2A shows 1 representative experiment out of 3 (n= 5-6 mice/group); p** = 0,0027 for DNP-challenged Aβ KO versus WT B6 (p** = 0,0074 and p = * 0,0286, for the other two experiments). Figure 2B data are from 2 pooled experiments (n= 5 mice/group); p* = 0,017 for DNP-challenged WT B6 versus anti-CD4-mAb treated mice and p* = 0,05 for DNP-challenged WT versus anti-CD25 mAb-treated B6 mice.

Article Snippet: C57BL/6 mice were injected intraperitonealy ( i.p ) with either 500 μg of the anti-CD25 mAb (clone PC61.5.3-Rat IgG1, BioXcell, USA) or anti-CD4 mAb (clone GK 1.5-Rat IgG2b, BioXcell) one day before sensitization with anti-DNP IgE.

Techniques: Injection, Control

Journal: PLoS ONE

Article Title: Regulatory CD4 + Foxp3 + T Cells Control the Severity of Anaphylaxis

doi: 10.1371/journal.pone.0069183

Figure Lengend Snippet: A : DEREG mice were injected with 1µg of DT ( triangles ) or with PBS ( squares ) on day -1, injected with anti-DNP IgE on day 0 and challenged with DNP-HSA ( black symbols ) or PBS ( white symbols ) on day +1. Body temperature was measured at various times after challenge. The data show pooled values from 3 experiments using 4-6 mice/group in each. p** = 0,0007 (2 way ANOVA) for DNP-challenged DEREG with and without DT (p values for each independent experiments are p= 0,032; p= 0,0176 and p=0,0105). B : plasma histamine ( left panel ) and serum mMCP-1 ( right panel ) were titrated in IgE-sensitized DEREG mice that were DT-untreated (black bars) or DT-treated ( grey bars ). IgE-sensitized and PBS-challenged DT-untreated DEREG mice ( white bars ) were used as negative controls. Data show mean ± SEM values (n= 8-10 mice/group) of each factor expressed as ng/ml. Statistical analysis by 2 way ANOVA comparing DEREG with and without DT show significance with p** = 0,0085. C : DT-untreated ( white symbol ) or DT-treated ( black symbols ) DEREG mice were either un-transferred ( black triangles ) or transferred with CD4 + Foxp3 + Treg from either naïve mice ( black diamond ) or from day 6 DNFB-sensitized mice ( black circles ). Mice were passively sensitized with IgE anti-DNP, challenged with DNP-HSA as indicated and body temperature was measured at various times after challenge. Data show mean (± SEM) of body temperature using 5 mice/group.

Article Snippet: C57BL/6 mice were injected intraperitonealy ( i.p ) with either 500 μg of the anti-CD25 mAb (clone PC61.5.3-Rat IgG1, BioXcell, USA) or anti-CD4 mAb (clone GK 1.5-Rat IgG2b, BioXcell) one day before sensitization with anti-DNP IgE.

Techniques: Injection, Clinical Proteomics