Journal: International Urogynecology Journal

Article Title: Local immune response in bladder pain syndrome/interstitial cystitis ESSIC type 3C

doi: 10.1007/s00192-013-2112-0

Figure Lengend Snippet: Statistics for gene expression (RT-qPCR)

Article Snippet: The following TaqMan® Gene Expression Assays (4331182) were analysed: cytotoxic T-lymphocyte-associated protein 4 ( CTLA4 , Hs03044418_m1) as a T-cell marker; CD20 (membrane-spanning 4-domains, subfamily A, member 1 MS4A1 , Hs00544819_m1) and CD79A molecule, immunoglobulin-associated alpha ( CD79A , Hs00233566_m1) as B-cell markers; immunoglobulin heavy locus ( IGH@ , Hs00378230_g1) as a marker for antibody heavy chain gene expression; and uroplakin 1B ( UPK1B , Hs00199583_m1), uroplakin 3A ( UPK3A , Hs00199590_m1) and cytokeratin 20 ( KRT20 , Hs00300643_m1) as urothelial markers.

Techniques: Gene Expression

Journal: International Urogynecology Journal

Article Title: Local immune response in bladder pain syndrome/interstitial cystitis ESSIC type 3C

doi: 10.1007/s00192-013-2112-0

Figure Lengend Snippet: Cut-off values and accuracy of biomarkers for BPS/IC ESSIC type 3C

Article Snippet: The following TaqMan® Gene Expression Assays (4331182) were analysed: cytotoxic T-lymphocyte-associated protein 4 ( CTLA4 , Hs03044418_m1) as a T-cell marker; CD20 (membrane-spanning 4-domains, subfamily A, member 1 MS4A1 , Hs00544819_m1) and CD79A molecule, immunoglobulin-associated alpha ( CD79A , Hs00233566_m1) as B-cell markers; immunoglobulin heavy locus ( IGH@ , Hs00378230_g1) as a marker for antibody heavy chain gene expression; and uroplakin 1B ( UPK1B , Hs00199583_m1), uroplakin 3A ( UPK3A , Hs00199590_m1) and cytokeratin 20 ( KRT20 , Hs00300643_m1) as urothelial markers.

Techniques:

Journal: Molecules (Basel, Switzerland)

Article Title: Inosine Prevents Colorectal Cancer Progression by Inducing M1 Phenotypic Polarization of Macrophages.

doi: 10.3390/molecules30010123

Figure Lengend Snippet: Figure 1. Inosine promoted macrophage polarization toward the M1 phenotype. (A) RAW264.7 cells were treated with inosine (0–40 mM) for 24 h; then, an MTT assay was performed to observe cell viability. RAW264.7 cells were administrated with inosine (1.25, 2.5, and 5 mM) in the absence or presence of LPS+IFN-γ or IL-4. (B,C) The proportions of CD86 and CD206-positive cells were determined by a flow cytometer. (D,E) The expression levels of CD86 and CD206 mRNA were detected by RT-qPCR. (F–H) Levels of CD86 and iNOS were measured by WB. Compared to the control group: * p < 0.05, ** p < 0.01, *** p < 0.001; Compared with the LPS+IFN-γ induced M1 group: # p < 0.05, ## p < 0.01, ### p < 0.001; Compared with the IL-4 induced M2 group: ∆∆∆p < 0.001.

Article Snippet: Primary antibodies against CD86 (1:1000, 26903-1-AP, Proteintech, Wuhan, China), iNOS (1:300, 22226-1-AP, Proteintech, Wuhan, China), β-actin (1:2000, GB15003-100, Servicebio, Wuhan, China) and secondary antibody against HRP Goat Anti-Rabbit IgG (1:5000, AS014, ABclonal, Wuhan, China) were used, respectively.

Techniques: MTT Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Control

Journal: Molecules (Basel, Switzerland)

Article Title: Inosine Prevents Colorectal Cancer Progression by Inducing M1 Phenotypic Polarization of Macrophages.

doi: 10.3390/molecules30010123

Figure Lengend Snippet: Figure 4. Effects of inosine on immune factors in the CT26 tumor microenvironment. (A) Effect of inosine on Ki-67 expression in tumor tissues (Scale: 100 µm; 400× and 200×). (B) Statistics of Ki-67 protein positive expression in tumor tissues (n = 3). (C) Fluorescence co-localization fluorogram of M1- type macrophage marker F4/80 + CD86 (scale: 100 µm; 200×). (D) F4/80 + CD86 expression statistics in tumor tissues. (E) M2 type macrophage marker F4/80 + CD206 fluorescence co-localization fluorogram (scale: 100 µm; 200×). (F) F4/80 + CD206 expression statistics in tumor tissues. Note: 5-Fu are 5-Fu (12 mg/kg) groups. IS-L and IS-H are inosine low and high-dose (5 mg/kg and 50 mg/kg) groups, respectively. Yellow arrows represent positive positions. Compared with the model group: ## p < 0.01; Compared with the 5-Fu group: △p < 0.05.

Article Snippet: Primary antibodies against CD86 (1:1000, 26903-1-AP, Proteintech, Wuhan, China), iNOS (1:300, 22226-1-AP, Proteintech, Wuhan, China), β-actin (1:2000, GB15003-100, Servicebio, Wuhan, China) and secondary antibody against HRP Goat Anti-Rabbit IgG (1:5000, AS014, ABclonal, Wuhan, China) were used, respectively.

Techniques: Expressing, Fluorescence, Marker

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Sequences of selected clones and their binding specificity. ( A ) Selected amino acid sequences of clones from PURE frex ® RD for CTLA-4-Fc. ( B ) Binding specificity of clones in ( A ) was evaluated as follows: mRNA–ribosome–peptide complex of each clone was reacted with each biotinylated protein (CTLA-4-Fc, dotted box; Human-Fc, black box; (-), white box) immobilized on streptavidin magnetic beads. Number of isolated mRNAs was determined by qPCR.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Clone Assay, Binding Assay, Magnetic Beads, Isolation

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Inhibition of interaction between CTLA-4 and B7-1 by C4-302. Inhibitory activity of C4-302 was examined. C4-351, which was found to bind to Fc region of CTLA-4, was used as negative control. After mixing mRNA–ribosome–peptide complex of each clone with CTLA-4, B7-1 immobilized on streptavidin magnetic beads via biotin was added to the mixture. ( A ) Because C4-351 bound to the Fc region of CTLA-4, mRNA was collected at nearly 10 10 . ( B ) In C4-302, mRNA collection was reduced considerably to just under 10 7 . This finding suggests that the C4-302 mRNA–ribosome–peptide complex inhibited the interaction of CTLA-4 with B7-1.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Inhibition, Activity Assay, Negative Control, Magnetic Beads

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Characterization of C4m-3127 synthetic peptide. Triangles indicate luminescence ( A ) and fluorescence ( B ) values for each concentration of C4m-3127 synthetic peptide. ( A ) Half-maximal inhibitory concentration (IC 50 ) value of synthetic cyclic peptide was measured using CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay Kit (BPS Bioscience Inc.) in 11-point two-fold serial dilution from 500 to 0.0050 μM by assay buffer. ( B ) Flow cytometry affinity analysis. CTLA-4 overexpressing CHO cell line was prepared at 1 × 10 5 cells/100 mL and reacted with synthetic peptide C4m-3127 with biotin-PEG4-Lys to N-terminus. Peptide in 10-point two-fold serial dilution from 4.4 to 0.0086 μM by PBS was used for this measurement. Cells with biotinylated peptide bound to their surface were detected with streptavidin–IFluor 647 conjugate. Fluorescence intensity depending on the amount of bound peptide shown on vertical axis.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Fluorescence, Concentration Assay, Screening Assay, Serial Dilution, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Sequence mimic: Strategy of transforming cyclic peptides to small molecules. ( A ) Conceptual diagram of interaction between C4m-3127 and CTLA-4. Dashed lines indicate hydrogen bonds. C, cysteine. Numbers from 1 to 12 correspond to sequence 1 MHPFLPIVSHHF 12 . ( B ) Chemical structure of β-turn. Dashed lines indicate hydrogen bonds. ( C ) Representative PepMetics ® scaffold used in this research, among more than 40 kinds of scaffolds. For synthetic reasons, PepMetics ® scaffold cannot mimic histidine at the 3-position and proline at 6- and 8-positions. ( D ) Conceptual diagram of sequence mimic, with 10 patterns for mimicking sequences from (1, 2, 3) to (10, 11, 12). Because of the limitation of PepMetics ® for proline mimicking, this scaffold did not mimic the four sequences (M1, H2, P3), (H2, P3, F4), (F4, L5, P6), and (L5, P6, I7).

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Sequencing

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Chemical structures of key compounds. IC 50 values show CTLA-4/B7-1 interaction inhibitory activity. Three amino acids (V10, H11, F12) on PGF00432 correspond to C4m-3127 H10V random loop: 1 MHPFLPIVSVHF 12 .

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Activity Assay

Journal: Pharmaceuticals

Article Title: Rational Strategy for Designing Peptidomimetic Small Molecules Based on Cyclic Peptides Targeting Protein–Protein Interaction between CTLA-4 and B7-1

doi: 10.3390/ph15121506

Figure Lengend Snippet: Predicted binding interaction of PGF00432 and PGF00506 with CTLA-4. ( A ) Crystal structure of B7-1/CTLA-4 complex (PDB code: 1I8L). Docking regions on CTLA-4 are derived from binding interface of the complex. ( B ) Predicted binding interaction of PGF00432 with CTLA-4. ( C ) Predicted binding interaction of PGF00506 with CTLA-4. Carbon, nitrogen, oxygen, and hydrogen are shown, respectively, in cyan (CTLA-4) or magenta (B7-1, PGF00432, and PGF00506), blue, red, and white. CTLA-4 and B7-1 are depicted as cyan and magenta ribbons, respectively. Dashed lines represent hydrogen bond interactions.

Article Snippet: Next, the inhibitory activity of the MBP–C4m-3127 peptide–His-tag fusion protein was tested using a kit (CTLA-4:B7-1[Biotinylated] Inhibitor Screening Assay; BPS Bioscience Inc. San Diego, CA, USA).

Techniques: Binding Assay, Derivative Assay