Journal: PLoS ONE

Article Title: Tissue Plasminogen Activator and Plasminogen Activator Inhibitor 1 Contribute to Sonic Hedgehog-Induced In Vitro Cerebral Angiogenesis

doi: 10.1371/journal.pone.0033444

Figure Lengend Snippet: Panels A to C show representative images of capillary-like tube formation of primary cerebral endothelial cells incubated with control medium (A), rhShh (B), and rhShh in presence of cyclopamine (C). Panel D shows quantitative data of capillary-like tube lengths in mm/mm 2 (n = 6/group). Real-time RT-PCR analysis (n = 3/group) showed that rhShh substantially increased mRNA levels of Ptch (E), a Shh receptor, and Gli 1(F), a transcription factor in endothelial cells. *p<0.05 the control group, # p<0.05 versus the rhShh group. Bar = 100 µm. WT = wild-type. CY = cyclopamine.

Article Snippet: MBECs were incubated with or without rhShh (100 ng/ml) (R&D System, Minneapolis, MN), cyclopamine (5 µM) (EMD-Calbiochem, Gibbstown, NJ), rhtPA (1 µM) (EMD-Calbiochem), or PAI-1 (10 ug/ml) (EMD-Calbiochem) for 16 h. MBECs (2×10 4 cells/well) were then seeded in the coated 96-well plates for 3–4 hours at 37°C.

Techniques: Incubation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Tissue Plasminogen Activator and Plasminogen Activator Inhibitor 1 Contribute to Sonic Hedgehog-Induced In Vitro Cerebral Angiogenesis

doi: 10.1371/journal.pone.0033444

Figure Lengend Snippet: Real-time RT-PCR (A, B) and Western blot (C to F) analyses showed that rhShh robustly increased Ang1 (A, C, D) and VEGF (B, E, F) expression in wild-type (WT) MBECs. Knockout of tPA (tPA−/−) substantially reduced Ang1 expression compared with wild-type MBECs (A, C, D), whereas incubation of tPA−/− MBECs with rhShh completely rescued Ang1 expression (A, C, D). However, incubation of tPA−/− MBECs with rhShh did not elevate VEGF expression, although knockout tPA significantly reduced VEGF protein levels (B, E, F). * p<0.05 versus the WT group, # p<0.05 versus the tPA−/− group. (n = 3/group). MBECs = mouse brain endothelial cells.

Article Snippet: MBECs were incubated with or without rhShh (100 ng/ml) (R&D System, Minneapolis, MN), cyclopamine (5 µM) (EMD-Calbiochem, Gibbstown, NJ), rhtPA (1 µM) (EMD-Calbiochem), or PAI-1 (10 ug/ml) (EMD-Calbiochem) for 16 h. MBECs (2×10 4 cells/well) were then seeded in the coated 96-well plates for 3–4 hours at 37°C.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Knock-Out, Incubation

Journal: eBioMedicine

Article Title: Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

doi: 10.1016/j.ebiom.2023.104826

Figure Lengend Snippet: Effect of multi-kinase inhibitor, checkpoint inhibitor (anti-PD1) and combination therapy on PCTS from patient 168: tumour slices had reduced cell proliferation in response to immunotherapy within 8 days of culture. Tissue slices from pt 168 were treated with doxorubicin (red), anti-PD-1 blocking antibody (blue), regorafenib (pink) and combination therapy (purple) and compared to untreated control (black). The following parameters were assessed in n PCTS ≥2 per timepoint. a | Cell proliferation, shown as percentage of Ki67 positive cells, in PCTS treated with the indicated drugs at the indicated time points; mean ± SD. b | Apoptosis, shown as percentage of TUNEL positive cells, in PCTS treated with the indicated drugs at the indicated timepoints; mean ± SD. Statistical test: 2-way ANOVA.

Article Snippet: PCTS were exposed to the following drugs and analysis was performed at the timepoints indicated in : doxorubicin (Doxo, Selleck chem, S1208), 2 μM , ; regorafenib (Selleck chem, S5077), 5 μM , ; nivolumab (Selleck chem, A2002), 2.6 nM ; anti-PD-1 blocking IgG (R&D, MAB10864), 0.46 μg/mL; ipilimumab (Selleck chem, A2001), 0.5 nM ; bevacizumab (Selleck chem, A2006), 0.5 nM ; atezolizumab (Selleck chem, A2004), 0.5 nM.

Techniques: Blocking Assay, TUNEL Assay

Journal: eBioMedicine

Article Title: Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing

doi: 10.1016/j.ebiom.2023.104826

Figure Lengend Snippet: Effect of multi-kinase inhibitor, checkpoint inhibitor (anti-PD1) and combination therapy on PCTS from patient 168: tumour slices respond to immunotherapy within 8 days of culture. Tissue slices from pt 168 were treated with doxorubicin (red), anti-PD-1 blocking antibody (blue), regorafenib (pink) and combination therapy (purple) and compared to untreated control (black). The following parameters were assessed in n PCTS ≥2 per timepoint. a | Weight-adjusted intracellular ATP levels of tissue slices. b | AEC of tissue slices from patient 168 treated with indicated treatment combinations. c | Representative images for H&E, haematoxylin + Sirius red, Ki67, TUNEL, CD45 (leucocytes) and CD3 (T-cells) for treated tissue slices. Scale bars: 100 μm, unless otherwise indicated.

Article Snippet: PCTS were exposed to the following drugs and analysis was performed at the timepoints indicated in : doxorubicin (Doxo, Selleck chem, S1208), 2 μM , ; regorafenib (Selleck chem, S5077), 5 μM , ; nivolumab (Selleck chem, A2002), 2.6 nM ; anti-PD-1 blocking IgG (R&D, MAB10864), 0.46 μg/mL; ipilimumab (Selleck chem, A2001), 0.5 nM ; bevacizumab (Selleck chem, A2006), 0.5 nM ; atezolizumab (Selleck chem, A2004), 0.5 nM.

Techniques: Blocking Assay, TUNEL Assay

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: The immunomodulatory lectin Gal-9 strongly binds naive and memory B cells but is inhibited in GC B cells by I-branching of N-glycans. a Representative histograms (left) and quantification (right) of recombinant Gal-9 (top) and Gal-1 (bottom) by flow cytometry to tonsillar naive, GC, and memory B cells ex vivo. Gray histogram represents staining in the presence of 100 mM lactose, a competitive inhibitor of galectin binding. b Schematic of reported I-branch activity of the glycosyltransferase GCNT2 on N-glycans. GCNT2 initiates I-branching via transfer of an N -acetylglucosamine in a β1,6 linkage, followed by subsequent galactosylation by β1,4 galactosyltransferases (β4GalTs). c Quantitative real-time reverse-transcription PCR (qRT-PCR) analysis of GCNT2 gene expression in human B cell subsets sorted as in Fig. and Supplementary Fig. . d Representative histograms (left) and quantification (right) of recombinant Gal-9 (top) and Gal-1 (bottom) binding by flow cytometry to control ( GFP -transduced) or GCNT2 -transduced overexpression variant NUDUL-1 B cells. Gray histograms represent Gal-1 or Gal-9 staining in the presence of 100 mM lactose. e Representative histograms (left) and quantification (right) of recombinant Gal-9 (top) and Gal-1 (bottom) binding by flow cytometry to Ramos B cells transduced with control shRNA (Scr) or shRNAs targeting GCNT2 . For a , n = 10, where each data point represents an individual tonsil specimen, pooled from two independent experiments. For c , n = 5 distinct tonsil specimens pooled from at least three independent experiments. For d and e , n = 3 biological replicates from three independent experiments. For a , c , and d statistics were calculated using one-way ANOVA with correction for multiple comparisons. For e , statistics were calculated using an unpaired, two-tailed t -test. Throughout, bars and error bars depict mean and standard error of the mean (SEM), respectively. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Recombinant, Flow Cytometry, Ex Vivo, Staining, Binding Assay, Activity Assay, Quantitative RT-PCR, Expressing, Over Expression, Variant Assay, Transduction, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: Gal-9 is expressed by naive B cells and binds the glycoprotein CD45. a In situ expression of Gal-9 in human tonsil. Formalin-fixed, paraffin-embedded tonsil sections were stained with Gal-9 antibody, PAX5 antibody (to identify B cells), or isotype control, followed by counterstaining with hematoxylin. Scale bar, 200 μm. b Relative comparison of Gal-9 expression by qRT-PCR of FACS-sorted naive, GC, memory B cells (as in Fig. and Supplementary Fig. ). Data were normalized to a housekeeping control gene VCP and are shown relative to naive B cells. c Representative western blot (left) quantification (right) of Gal-9 protein expression in lysates of FACS-sorted B cell subsets. Data were normalized to β-actin to control for differences in loading. For Gal-9 blot, the doublet band indicates expression of both medium-length and full-length Gal-9 isoforms. d Representative histogram (left) and quantification (right) of endogenous cell surface Gal-9 detected by ex vivo flow cytometry of unpermeabilized naive, GC, and memory B cells. e Co-immunoprecipitation of Gal-9 and CD45 from naive B cell lysates. Untouched naive B cells were magnetically enriched, labeled with Gal-9 (2 μg mL −1 ), lysed, and immunoprecipitated/blotted with the indicated antibodies. f Representative immunofluorescence images of CD45 capping following Gal-9 treatment (4 μg mL −1 ). Scale bar, 10 μm. For a , data are representative of similar results from n = 3 distinct tonsil specimens. For b , n = 4 separate tonsil specimens pooled from more than three independent experiments. For c , n = 3 distinct tonsil specimens pooled from three independent experiments. For d , n = 10, where each data point represents a disparate tonsil specimen pooled from two independent experiments. For e and f , data are representative of three independent experiments using three distinct tonsil specimens. For b and d , statistics were calculated using one-way ANOVA with correction for multiple comparisons. For c , statistics were calculated using a two-tailed, one sample t -test against a hypothetical value of 1. Throughout, bars and error bars depict mean and SEM, respectively. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: In Situ, Expressing, Formalin-fixed Paraffin-Embedded, Staining, Quantitative RT-PCR, Western Blot, Ex Vivo, Flow Cytometry, Immunoprecipitation, Labeling, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: Gal-9 induces phosphorylation of Lyn, CD22, and SHP-1 in naive B cells. a Representative western blot and b quantification of Lyn, CD22, or SHP-1 phosphorylation at the indicated sites in magnetically enriched naive B cells treated with recombinant Gal-9 at the indicated concentrations in the absence (left blot) or presence (right blot) of anti-IgM F(ab’) 2 -mediated crosslinking (15 μg mL −1 ) for 5 min. Blots were subsequently stripped and reprobed with antibody against total protein. Data in b were normalized to respective total protein control and are presented relative to no stimulation control. In all experiments, no IgM and IgM-stimulation lanes were loaded on the same gel and probed on the same blot. For a , data are representative of results from four independent experiments. For b , n = 3 or more distinct tonsil specimens pooled from three or more independent experiments. Statistics were calculated using a one-sample t -test against a hypothetical value of 1. Throughout, bars and error bars depict mean and SEM, respectively. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Western Blot, Recombinant

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: Gal-9 suppresses BCR-mediated calcium signaling and nuclear translocation of NFAT1. a Flow cytometric measurement of B cell cytoplasmic calcium levels using Fluo-4AM indicator dye following anti-BCR F(ab’) 2 treatment (20 μg mL −1 ) in the presence or absence of recombinant Gal-9 (2.5 μg mL −1 ) and/or lactose (25 mM). Data shown are gated on CD19 + CD44 hi B cells. Mean fluorescence intensities (MFI) were normalized to average MFI of 30 s baseline. Arrow, time of stimulus. b Area under the curve (AUC) and c peak MFI of data presented in a . d Representative western blot and e quantification of NFAT1 nuclear translocation in magnetically enriched naive B cells treated with anti-IgM F(ab’) 2 (15 μg mL −1 ) in the presence or absence of recombinant Gal-9 (2 μg mL −1 ) before lysis, fractionation, and blotting with NFAT1 Ab. As controls, cells were incubated in lactose (25 mM), cyclosporin A (100 ng mL −1 ), or ionomycin (1 μM). Blots were reprobed for β-tubulin and Histone H3 as loading and fractionation controls. f Representative immunocytochemistry and g quantification of NFAT1 nuclear translocation in naive B cells treated as in d and e . Scale bar, 10 μm. h Measurement of calcium flux in Arthrobacter ureafaciens sialidase-treated primary B cells, following the indicated stimulus. For a and h , solid line represents mean of five ( a ) or three ( h ) tonsil specimens from the same number of experiments. For b and c , n = 5, where each data point represents a distinct specimen pooled from five experiments. For d , data are representative of four experiments. For e , n = 4 specimens pooled from four experiments (CsA and ionomycin, n = 1). For f , data are representative of two experiments. For g , each data point represents average localization score for a single microscopic field (binary scoring of 1 = cytoplasmic or −1 = nuclear). Results are representative of two experiments. For b , c , and e , statistics were calculated using one-way ANOVA with correction for multiple comparisons. For g , a Kruskal–Wallis test was used with Dunn’s correction for multiple comparisons. For h , statistics were calculated using a two-way repeated measures ANOVA using factors “time” and “treatment,” with Bonferroni correction for multiple comparisons. Throughout, bars and error bars depict mean and SEM. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Translocation Assay, Recombinant, Fluorescence, Western Blot, Lysis, Fractionation, Incubation, Immunocytochemistry

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: Gal-9 inhibits B cell activation and proliferation. a Representative contour plots and b quantification of naive B cell proliferation 40 h post-activation with either T-independent (anti-IgM F(ab’) 2 + unmethylated CpG oligonucleotides + IL-2/4/10) or T-dependent stimulation (anti-IgM F(ab’) 2 + recombinant CD40L-trimer + IL-2/4/10/21), in the presence or absence of the indicated concentration of recombinant Gal-9. “4 + L” indicates 4 μg Gal-9 plus 10 mM lactose; “4 + S” indicates 4 μg Gal-9 plus 10 mM sucrose, a non-inhibitory osmolarity control. c Representative histograms and d quantification of CD86 (B7-2) expression on naive B cells 40 h after activation in the presence or absence of Gal-9 at the indicated concentrations, as in b . For a and c , data are representative of five independent experiments. For b and d , n = 5 distinct tonsil specimens pooled from five independent experiments. For b , and d , statistics were calculated using a one-sample t -test against a hypothetical value of 100. Throughout, bars and error bars depict mean and SEM, respectively. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Activation Assay, Recombinant, Concentration Assay, Expressing

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: B cell-intrinsic Gal-9 is a negative regulator of BCR calcium signaling. a Flow cytometric assessment of B cell cytoplasmic calcium levels using Fluo-4AM indicator dye following anti-BCR F(ab’) 2 stimulation (20 μg mL −1 ) after 90 min culture in the presence or absence of 25 mM lactose. Depicted are results from CD44 hi CD19 + B cells. Mean fluorescence intensities (MFI) over the entire acquisition period were normalized to the average MFI of the 30 s baseline. Arrow, time of stimulus. b Western blot of lysates from indicated primary cells or B cell lines with the specified combination of Abs. c Analysis of calcium flux following anti-BCR F(ab’) 2 stimulation (20 μg mL −1 ) in Karpas 1718 B cells (CD22 + and SHP-1 + ) transduced with either control shRNA (Scr) or one of two shRNAs against LGALS9 . d Pre-stimulation baseline, e peak, or f total calcium flux from experiments presented in c . g Analysis of calcium flux following anti-BCR stimulation (20 μg mL −1 ) in NUDUL-1 B cells (CD22 lo and SHP-1 lo ) transduced with either control shRNA (Scr) or one of two shRNAs against LGALS9 . h Pre-stimulation baseline, i peak, or j total intracellular calcium levels from experiments presented in g . k Analysis of calcium flux following anti-BCR stimulation (20 μg mL −1 ) as in c , except that 10 μg mL −1 recombinant Gal-9 was co-administered with BCR stimulus where indicated. l Peak or m total intracellular calcium levels from experiments presented in k . For a , c , g , and k , the solid line represents the mean and shaded error bars represent SEM from five ( a ), four ( c and g ), or six ( k ) different biological replicates pooled from the same number of independent experiments. For d – f and h – j , n = 4, where data points depict biological replicates pooled from four independent experiments. For l – m , n = 6, where data points depict biological replicates pooled from six independent experiments. For a , statistics were calculated using a two-way repeated measures ANOVA using factors “time” and “treatment,” with Bonferroni correction for multiple comparisons. For d – f , h – j , and l – m , statistics were calculated using one-way ANOVA with correction for multiple comparisons. Throughout, bars and error bars depict mean and SEM, respectively. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Fluorescence, Western Blot, Transduction, shRNA, Recombinant

Journal: Nature Communications

Article Title: Galectin-9 suppresses B cell receptor signaling and is regulated by I-branching of N-glycans

doi: 10.1038/s41467-018-05770-9

Figure Lengend Snippet: Proposed role of Gal-9 and I-branches in the regulation of BCR signaling in naive B cells vs. GC B cells. In naive B cells (right), Gal-9 is expressed at high levels and secreted into the microenvironment by an unconventional mechanism. Autologously, or exogenously, produced Gal-9 binds poly-LacNAc-containing N-glycans on CD45, which are predominantly of the linear type due to low expression of the I-branching glycosyltransferase GCNT2. Gal-9 binding to CD45 activates Lyn (by an undetermined mechanism), which subsequently phosphorylates tyrosine residues in CD22 ITIMs and recruits the protein tyrosine phosphatase SHP-1. SHP-1 phosphatase activity dampens cytoplasmic calcium levels, including calcium accumulation in response to BCR engagement, possibly through its reported ability to activate B cell calcium efflux pumps. Diminished intracellular calcium levels results in decreased nuclear translocation and activity of NFAT1 and other calcium sensitive signaling factors, ultimately inhibiting B cell activation. By contrast, Gal-9 activity is downmodulated in GC B cells (left) via the combined downregulation of Gal-9 protein and upregulation of GCNT2 , which disfavors Gal-9 binding by modifying N-glycan poly-LacNAcs with I-branches

Article Snippet: For Gal-9 IP, cells were treated with 2 μg mL −1 recombinant Gal-9 (R&D) in 1% BSA for 30 min on ice, followed by washing and lysis in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA).

Techniques: Produced, Expressing, Binding Assay, Activity Assay, Translocation Assay, Activation Assay

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 1 week in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) 7–9 days before harvest (n = 5 pairs from four experiments). (A–D) Response curves. Restriction response to various concentrations of PGF2α (A) and relaxation response curves for nitroprusside (B), bradykinin (C), or substance P (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. mN, milliNewtons. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD45, and HLA-DR. The staining for human CD45 is indistinguishable from that for human CD3 (data not shown). Original magnification, ×200.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 2 weeks in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) for 13–15 days (n = 4–7 pairs from four to six experiments). (A–D) Response curves. Constriction response to various concentrations of PGF2α (A) and relaxation response to bradykinin (B), nitroprusside (C), or YC-1 (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD3, and iNOS. iNOS expression was highly localized to infiltrating T cells (arrows). Original magnification: CD31 and CD3 panels, ×200; iNOS panels, ×400.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Expressing

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of IFN-γ neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti–IFN-γ mAb (filled triangles) or control mAb (IgG2a) (open triangles) for 2 weeks (n = 5 matched triplicates from three experiments). (A and B) Endothelium-independent responses to various concentrations of PGF2α (A) and nitroprusside (B). (C and D) Endothelium-dependent response to bradykinin before (C) and after (D) treatment with L-NAME. *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG2a group, n = 3–5. (E) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. The luminal HLA-DR–positive cells are also human CD31–positive (data not shown). Goat anti-mouse secondary Ab alone did not stain the grafts. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (F and G) RNA transcript levels of iNOS (F) and eNOS (G) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Quantitative RT-PCR

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of TNF neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti-TNF mAb (filled triangles) or control mAb (IgG1) (open triangles) for 2 weeks (n = 4 matched triplicates from two experiments). (A–C) Concentration-dependent responses to PGF2α (A), nitroprusside (B), and bradykinin (C). *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG1 group; n = 3–4. (D) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (E and F) RNA transcript levels of iNOS (E) and eNOS (F) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Concentration Assay, Immunohistochemistry, Staining, Quantitative RT-PCR