Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: Heat shock protein 60 ( HSP 60) induces dendritic cell ( DC ) activation/maturation; 1 of 3 individual experiments is presented here. DC s at the concentration of 2×10 6 /mL were cultured with or without HSP 60. A, Expression of CD 86, CD 83, CD 40, and HLA ‐ II was analyzed by flow cytometry after 24 h of stimulation with 5 μg/mL of HSP 60. Figure in histogram shows higher induction of these markers by HSP 60. Percentage of histogram shift or mean fluorescence intensity was calculated from triplicate samples and P values are CD 86 ≤0.001, CD 83 ≤0.001, HLA ‐ II ≤0.05, and CD 40 ≤0.001. B, Cytokine profile of DC against HSP 60 is listed. DC s were stimulated as mentioned, cultured for 24 h, and cell supernatant was collected for measurement of cytokines. Mostly pro‐inflammatory cytokines are highly increased by HSP 60. C, In similar condition, DC s activation was observed by HSP 90 (5 μg/mL). P value from triplicates samples CD 86 ≤0.0001, CD 83 ≤0.001, CD 40 ≤0.01, and HLA ‐ II ≤0.001. D, HSP 90 induced‐ DC s were cocultured with T cells but no activation of T cells was observed. APC indicates antigen‐presenting cells; FITC, fluorescein isothiocyanate; IL‐6, interleukin‐6; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5; TGF‐β1, transforming growth factor‐β1.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Activation Assay, Concentration Assay, Cell Culture, Expressing, Flow Cytometry, Fluorescence

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: T‐cell activation and proliferation in dendritic cell ( DC )+T‐cell coculture. A, DC s were stimulated with heat shock protein 60 ( HSP 60) at the concentration of 2.5, 5, or 10 μg/mL. After overnight incubation, autologous T cells 4×10 5 were cocultured with 1×10 5 DC s. All the concentrations of HSP 60 induced T‐cell activation, where 5 or 10 μg/mL were a little stronger, which was tested by CD 25 expression in CD 3 T cells. B, One representative of minimum 3 experiments of T‐cell activation, which was determined by CD 69 early activation, CD 25 and CD 71 intermediate/late activation markers. DC s were stimulated with 5 μg/mL of HSP 60 and cocultured with CD 3 + T cells. For analysis, CD 3 + cells were gated, then percentage of CD 3+ CD 69/ CD 25/ CD 71 + cells was shown in the upper right of each gate. HSP 60‐induced DC s activated all of 3 activation markers in CD 3 + T cells, P ≤0.0001 from triplicate samples. C, In response to HSP 60, DC +T cells show a high proliferation rate; 1 representative of 3 individual experiments is shown here. APC Allophycocyanine; BrDu, 5‐brom‐2‐deoxiuridin; OD, Optical density; Percp‐Cy5.5, Peridinin Chlorophyll Protein‐Cyanine 5.5. * P ≤0.05; *** P ≤0.0001.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Activation Assay, Concentration Assay, Incubation, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: Plaque T‐cell activation. To investigate the plaque T‐cell activation, dendritic cells ( DC s) were generated from blood monocytes of atherosclerotic patients; DC s were stimulated with 5 μg/mL of heat shock protein 60 ( HSP 60) and coculture with the plaque T cells of the same patients. First, we have determined classic T‐cell activation marker CD 25 (intermediate activation) from 6 patients, where we observed CD 25 induction against HSP 60. Next, early activation marker CD 69 and late activation marker CD 71 from 4 patients also tested against HSP 60. The induction of CD 69 and CD 71 also was similar to CD 25 induction. Cells were cultured in triplicates (with determination of statistical significance) or duplicates (where material from plaques was not sufficient for triplicates) from each patient. * P ≤0.05; ** P ≤0.01; *** P ≤0.0001.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Activation Assay, Generated, Marker, Cell Culture

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: T helper (Th) cells differentiation: 1 representative experiment of 3 individual experiments. A, Heat shock protein 60 ( HSP 60) (5 μg/mL) induced DC s were cocultured with CD 3 + T cells. Cytokines were measured from coculture supernatants. In HSP 60 treated coculture, high level of pro‐inflammatory cytokines was observed. B, Next, Th cell differentiating transcription factors were analyzed by reverse transcriptase quantitative polymerase chain reaction. Th1 transcription factor T‐bet was increased more than 3‐fold whereas Th2 transcription factor GATA ‐3 increase was negligible. In addition, FoxP3 and RORC transcription factor for T regulatory cells and IL ‐17 producing T cells, respectively, also increased. C, Cell culture supernatants from the patients were used only for IFN‐γ measurement. IFN ‐γ detection from 4 patients shows higher induction by HSP 60. Patients’ samples were in duplicates. D, mRNA level or transcription factor was analyzed from 3 patients, where mostly Th1 transcription factor was increased. DC s indicates dendritic cells; FoxP3, Forkhead box P3; GATA3, GATA binding protein 3; IFN ‐γ, interferon‐γ; IL‐6, interleukin‐6; ns, not significant; RORC, RAR Related Orphan Receptor C; T‐bet, T‐box expressed in‐T‐cells; TNF‐α, tumor necrosis factor α; TGF‐β1, transforming growth factor‐β1. * P ≤0.05; ** P ≤0.01; *** P ≤0.0001.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Cell Culture, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: Heat shock protein 60 ( HSP 60; 5 μg/mL)‐induced dendritic cells ( DC s) were cocultured with T cells in 24‐well trans‐well culture plates. T cells 4×10 5 (upper well) in 200 μL were cultured in the upper chamber while autologous HSP 60‐stimulated DC s 2×10 5 (bottom well) in 500 μL were in the bottom chamber. In parallel, DC s induced in the same condition were cocultured with T cell in same well as mix coculture. T‐cell activation from trans‐well culture was weak in comparison to mix culture. One of 2 individual experiments is represented here (A). HSP 60‐induced DC s were cocultured with CD 3 + T cells in presence or absence of 8 μg/mL anti‐ HLA II antibodies ( HLA ‐ II ‐blocking antibodies). DC s‐mediated T‐cell activation was inhibited in presence of HLA II antibodies (B). Experiments were performed 3 times and 1 of them is shown as a representative, except for CD 69 where inhibition by HLA ‐ II ‐blocking antibodies was nonsignificant in some experiments. * P ≤0.05; ** P ≤0.01; *** P ≤0.0001.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Cell Culture, Activation Assay, Comparison, Blocking Assay, Inhibition

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

doi: 10.1161/JAHA.117.006778

Figure Lengend Snippet: Annexin A5 ( ANX A5) inhibited heat shock protein 60 ( HSP 60)‐induced immune response. Dendritic cells ( DC s) at the concentration of 2×10 6 were stimulated with HSP 60: 5 μg/mL in presence or absence of ANX A5 (10 μg/mL) and cocultured with CD 3 + T cells ( DC : T‐cell concentration was same as mentioned in Figure ). ANX A5 attenuated HSP 60‐induced T‐cell activation. One of 3 individual experiments is represented (A). HSP 60 induced P38 MAPK activation, mean fluorescence intensity was calculated and P ≤0.05 (C) but there was no effect on NF κB (B) activation. ANX A5 did not induce T regulatory cells (T‐reg cells) in HSP 60‐stimulated DC +T‐cell coculture, whereas little induction of T‐reg by HSP 60 was observed. The experiments were performed 3 times and 1 of them is presented (D). FoxP3‐positive CD 4 T cells did not show significant increase in response to ANX A5 or HSP 60. One of 2 individual experiments is shown (E). Protein–protein interaction assay shows HSP 60‐ ANX A5 have transient or weak interaction where HSP 60‐ ANXA 5 incubation reduced HSP 60 detection in ELISA measurement. One of 3 individual experiments is presented. F, Ox‐ LDL induced HSP 60 production in DC s and it was inhibited by ANX A5 in both protein (1 representative experiment from 3 individual experiments) (G) and mRNA level (H). Foxp3, Forkhead box P3; MAPK, Mitogen activated protein kinase; Ox‐LDL, oxidized low‐density lipoprotein; NF κB, nuclear factor kappa B; PE, Phicoerythrin; PE‐Cy7, Phicoerythrin‐cyanine 7. * P ≤0.05; ** P ≤0.01; *** P ≤0.0001.

Article Snippet: After 6 hours of culture condition, cell supernatant was collected and HSP60 was measured by human HSP60 Duoset ELISA kit (R&D systems, UK).

Techniques: Concentration Assay, Activation Assay, Fluorescence, Protein Protein Interaction Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Combined Therapy Versus Fortified Anti-VEGF Monotherapy in Type C Polypoidal Choroidal Vasculopathy: Long-Term Outcomes and Exploratory Biomarker Insights †

doi: 10.3390/ijms27031224

Figure Lengend Snippet: Various vascular markers for type C PCV, CSCR, and nvAMD. Before intravitreous injection (IVI), aqueous protein levels of VEGF/PlGF/HIF-1α/β-catenin/Wnt1 were detected using ELISA on patients with AMD, PCV, or CSCR. In ( A ), the PCV ( n = 17) or nvAMD ( n = 41; control) levels of VEGF were median [Q1, Q3]: 106.19 [80.00, 203.81] or 191.91 [116.68, 289.96]. In ( B ), the PCV ( n = 13) or nvAMD ( n = 29) levels of PlGF were median [Q1, Q3]: 1.39 [0.72, 18.45] or 15.48 [11.25, 17.90]. The PCV/CSCR/nvAMD (pre-IVI; n = 9/4/16) levels of β-catenin ( C ) were 983.77 ± 329.68/1224.33 ± 743.69/3622.41 ± 499.70. The PCV/CSCR/nvAMD (pre-IVI; n = 5/4/20) levels of HIF-1α ( D ) were median [Q1, Q3]: 6096.66/8705.06/1091.28 [5173.83/5939.04/803.69, 7811.37/14,239.77/3210.46]. Additionally, the PCV/CSCR (pre-IVI) levels of Wnt1 ( E ) were 1180.66 ± 172.41 ( n = 6)/1387.12 ± 289.54 ( n = 4). Significance was * p < 0.05.

Article Snippet: Protein levels in aqueous humor were quantified using commercial ELISA kits [ , ]: VEGF (BMS227/2, Bender MedSystems, Wien, Austria), PlGF (CBS-E07400r, Cusabio, Houston, TX 77054, USA), HIF-1α (M00013-2, Uscn Life Science, San Jose, CA 95123, USA), β-catenin (DYC1329, R&D Systems, Minneapolis, MN 55413, USA), and Wnt1 (RHF128CK, Antigenix America, Huntington Station, NY 11746, USA) [ ].

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control

Journal: bioRxiv

Article Title: Developing single molecule methods for measuring the pathway proteins ERK, AKT, cyclin d and p70s6k in localized colon cancer in relation to mutation status

doi: 10.1101/695809

Figure Lengend Snippet: The average number of enzyme per bead (AEB) against concentration is shown. The calibrators (n=8) were run in duplicate and the mean value for each point of the calibrators is shown.

Article Snippet: The biotinylated detector antibodies and the calibrators were tAKT (DYC1775), pAKT (DYC887B), tERK (DYC1230C), pERK (DYC1018B) (R&D Systems), pp70s6k (DYC896) and cyclin d (ab218793) (abcam).

Techniques: Concentration Assay