Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 1. Oncostatin M (OSM)–induced expression of CCL13. A and B, Normal dermal fibro- blasts (nDF), normal lung fibroblasts (nLF), normal cervical fibroblasts (nCF), and normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM for the indicated periods of time. A, Levels of CCL2, CCL13, and CCL11 mRNA were determined by RNase protection assay. Results are representative of 3 independent experiments. B, The amounts of CCL13 and CCL2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 3). P 0.05 versus control. C, Primary human blood neutrophils were stimulated with 10 ng/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) for 3 hours. The amounts of OSM in culture supernatants were determined by ELISA. Values are the mean SD (n 3). P 0.05 versus control. D, Culture supernatants (SNs) from untreated neutrophils or neutrophils stimulated with GM-CSF for 3 hours were transferred onto normal SFs. The contribution of OSM was evaluated by additional application of blocking antibodies to OSM or control antibodies (ctrl. Ab). The amounts of CCL13 produced in normal SF culture supernatants were then determined by ELISA. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus antibody control.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Rnase Protection Assay, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Produced

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 2. Significance of OSM and other key cytokines involved in rheumatoid arthritis (RA) pathogenesis for CCL13 expression. A, Normal SFs were treated for 2 hours with 10 ng/ml of OSM, 200 units/ml of interleukin-6 (IL-6)/0.5 g soluble IL-6 receptor (sIL-6R), 10 ng/ml of IL-1, or 10 ng/ml of tumor necrosis factor (TNF). CCL13 mRNA levels were quantified, normalized to L32 levels, and compared with untreated cells. Values are the mean SD (n 4). P 0.05 versus specific control. B, Normal SFs were stimulated as described in A for 2 hours or 12 hours. Values are the mean SD (n 4). P 0.05 versus time-specific control. C, Normal SFs were treated for 3 hours with OSM, alone or in combination with IL-6/sIL-6R, IL-1, or TNF (at the concentrations described in A). Values are the mean SD (n 4). P 0.05 versus control. D, RASFs were stimulated with cytokines as described in A for 3 hours. Values are the mean SD (n 4). P 0.05 versus normal SF control; d P 0.05 versus control. E, OSM and CCL13 protein in the supernatant of unstimulated fibroblasts was determined by ELISA. Values are the mean SD (n 3). P 0.05 versus normal SF. F and G, Both normal SF and RASF supernatants were treated with OSM and either control or OSM-neutralization antibodies. Normal SFs and RASFs were restimulated with OSM-depleted supernatant for 3 hours. Values are the mean SD (n 3). and # P 0.05 versus control; d P 0.05 versus stimulated control. H, Supernatants of untreated RASFs were treated with control antibody, CCL13 antibody, or both CCL13 and OSM-neutralizing antibodies. Depleted supernatants were added to untreated normal SFs. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus control supernatant; # P 0.05 versus CCL13-depleted supernatant. w/o without; OASF osteoarthritis SF (see Figure 1 for other definitions).

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 4. Comparison of oncostatin M (OSM)–, interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R)–, IL-1–, and tumor necrosis factor (TNF)–induced signal transduction, showing a potential role of phosphorylated (pY) STAT-5 in CCL13 induction. A, Normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM, 200 units/ml of IL-6/0.5 g of sIL-6R, 10 ng/ml of IL-1, or 10 ng/ml of TNF. Lysates were subjected to Western blot analysis, using antibodies specific for the indicated proteins. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status or with loading control antibodies. B, Normal SFs were stimulated with OSM as indicated. Western blots of lysates were stained with the antibodies indicated. C, Normal SFs, normal cervical fibroblasts (nCF), normal lung fibroblasts (nLF), and normal dermal fibroblasts (nDF) were stimulated with 10 ng/ml of OSM. Cell lysates were analyzed as described in B. D, Normal SFs were transfected with control or STAT-5 small interfering RNA (siSTAT5). Forty-eight hours later, cells were stimulated with 10 ng/ml of OSM for 3 hours. CCL13 and CCL2 protein in normal SF culture supernatants was measured by enzyme-linked immunosorbent assay. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus stimulated siRNA control; w/o without.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Comparison, Transduction, Western Blot, Activation Assay, Control, Staining, Transfection, Small Interfering RNA, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 5. Role of OSM-induced p38 activation in the stabilization of CCL13 mRNA through inhibition of tristetraprolin (TTP). A and C, Normal SFs and rheumatoid arthritis SFs (RASFs), respectively, were pretreated with 10 M AG490, 10 M SB202190, 10 M U0126, 10 M SP600125, 1 M JAK inhibitor I (JI-1), or the solvent DMSO, and then exposed to 10 ng/ml of OSM for 3 hours. Subsequently, CCL13 protein was quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 4). and # P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. B, Western blots from treated normal SFs were analyzed using the indicated antibodies. D, Normal SFs were stimulated with 10 ng/ml of OSM for 30 minutes. Then, cells were washed and cultivated for an additional 20 minutes in OSM-free medium containing actinomycin D (4 M). SB202190 (10 M) was added to the medium before cells were restimulated with OSM for the indicated periods of time. CCL13 and GAPDH mRNA levels were analyzed by reverse transcription–polymerase chain reaction. One of 3 representative independent experiments is shown. E, Normal SFs were transfected with TTP or control siRNA. Forty-eight hours later, cells were pretreated with 10 M SB202190 or DMSO for 20 minutes and then stimulated with 10 ng/ml of OSM for 3 hours. CCL13 was measured by ELISA. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. See Figure 4 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Activation Assay, Inhibition, Solvent, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Transfection

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 6. Contribution of STAT-5 and ERK-1/2 to CCL13 expres- sion. RASFs were transfected with STAT-5 or control small interfering RNA (siRNA). Forty-eight hours later, cells were preincubated with DMSO or U0126 for 30 minutes, prior to stimulation with 10 ng/ml of OSM for 3 hours. CCL13 protein was measured in culture superna- tants of RASFs, by ELISA. Values are the mean SD (n 4). P 0.05 versus unstimulated control; # P 0.05 versus OSM- stimulated DMSO control; d P 0.05 versus stimulated siRNA control. Inset, Knockdown efficiency was controlled by Western blot analysis of lysates, using a specific antiserum against tyrosine- phosphorylated STAT-5 (pY-STAT5). After stripping, the blots were reprobed with antiserum recognizing STAT-3. See Figure 5 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Transfection, Control, Small Interfering RNA, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Stripping Membranes

Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Effect of VEGF on endothelial cell chemokine expression. (a–d) Confluent cultures of human endothelial cells were treated for 4 hours (a and c) or as a time course (b and d) with VEGF alone or VEGF in combination with IFN-γ as indicated. Total RNA was harvested from endothelial cells and the expression of chemokines was analyzed by RNase protection assay. Concentration- (a) and time-dependent (b) effects of VEGF on chemokine expression. (c and d) Effect of IFN-γ alone or in combination with VEGF on chemokine expression. Note that treatment with IFN-γ alone resulted in IP-10 expression (c, and d lane 7), and a combination of IFN-γ with VEGF resulted in a synergistic induction of IP-10 (c, and d lanes 2–6). Bar graphs to the right of each blot represent the quantitative analysis of IP-10 mRNA expression in three representative RNase protection assays as illustrated in c and d. (e) The production of IP-10 by ELISA in culture supernatants of endothelial cells treated with IFN-γ (1,000 U/ml; black bars) or with VEGF (10 ng/ml) and IFN-γ (1,000 U/ml) (white bars) for different times as indicated. Representative of three similar experiments performed in triplicate (mean ± 1 SD).

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Rnase Protection Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Function of VEGF in alloimmune T cell activation and allograft rejection. (a) Anti–human VEGF or anti–murine VEGF antiserum was added into the human or the mouse MLR, respectively. Proliferation was assessed by [3H]thymidine incorporation for the last 18 hours of coculture. (b) The production of IFN-γ and IL-2 was assessed by ELISA in coculture supernatants from a human MLR. As illustrated, blockade of VEGF had no effect on proliferation or cytokine production in the MLR. Bars indicate the mean ± 1 SD for triplicate wells. Data are representative of three experiments with similar results. S, stimulators alone; R, responders alone. (c) Frequency of IFN-γ-producing cells in murine recipients of cardiac transplants as assessed by ELISPOT. Illust production of IFN-γ from a syngeneic, an untreated, and an anti-VEGF-treated animal. Representative of three such experiments performed in triplicate.

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Function of VEGF-dependent regulation of IP-10 in allograft rejection. (a) Confluent cultures of murine myocardial endothelial cells were treated for 4 hours with recombinant murine VEGF or IFN-γ alone or VEGF in combination with IFN-γ as indicated. Total RNA was harvested from endothelial cells and the expression of IP-10 was analyzed by RNase protection assay. (b and c) The ability of VEGF to mediate IP-10-dependent trafficking and rejection was evaluated using anti-VEGF and anti–IP-10 in fully MHC-mismatched C57BL/6 (H-2b) or IP-10–/– (H-2b) donor hearts transplanted into BALB/c (H-2d) mice. Recipients of wild-type grafts were treated with anti-VEGF alone, or with anti-VEGF in combination with anti–IP-10. Recipients of IP-10–/– donor grafts were treated with anti-VEGF. Both anti–IP-10 and anti-VEGF were administered according to the schedule outlined in Methods. As illustrated in b, we found that addition of anti-VEGF with anti–IP-10 significantly prolonged allograft survival in wild-type combinations (P < 0.005); and in c, anti-VEGF prolonged survival in mice that received IP-10–/– donor hearts (P < 0.04). The survival of control untreated wild-type grafts are illustrated by the dotted line.

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Recombinant, Expressing, Rnase Protection Assay

Journal: Physiological Reports

Article Title: Salivary vascular growth factor responses to prolonged and interrupted sitting in young, healthy adults

doi: 10.14814/phy2.70798

Figure Lengend Snippet: Changes in salivary growth factor concentrations during prolonged sitting (PS), low‐intensity (LI), and moderate‐intensity (MI) interruption sessions. Box‐and‐whisker plots display pre‐ (Pre, 0 h) and post‐session (Post, 4 h) concentrations for (a) VEGF, (b) EGF, and (c) angiogenin. Panel (d) shows pre‐post percent change for all three biomarkers. The line within each box represents the median; whiskers indicate minimum and maximum values. VEGF decreased significantly in LI and MI but not PS; EGF showed no significant changes; and angiogenin increased significantly in all conditions. No significant differences were observed between conditions in the magnitude of pre‐post change.

Article Snippet: Samples were then analyzed using sandwich Enzyme‐Linked Immunoabsorbent Assays (ELISA) to examine EGF (Human EGF DuoSet ELISA, R&D Systems Inc.), VEGF (Human VEGF DuoSet ELISA, R&D Systems Inc.), and angiogenin (Human Angiogenin DuoSet ELISA, R&D Systems Inc.) following the manufacturer's protocol with absorbance measured at 450 nm using a Cytation 5 microplate reader (Bio‐Tek Instrumentation, VT, USA).

Techniques: Whisker Assay

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: Baseline characteristics of the participants in the pharmacokinetic study (n = 44) according to the predicted CYP2D6 phenotypes and CYP2D6 activity scores.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques: Activity Assay

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: Frequency of the CYP2D6 alleles, genotypes, phenotypes and activity scores.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques: Activity Assay

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: Pharmacokinetic parameters of primaquine and 5,6-orthoquinone primaquine in plasma and urine according to the predicted CYP2D6 phenotypes.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques: Clinical Proteomics

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: Plasma primaquine concentration time profiles according to the predicted CYP2D6 phenotypes (A) and the CYP2D6 activity scores (B) Data are presented in median ± median absolute deviation (MAD). MAD was calculated as median ( | X i - X ‾ | ) where X ‾ = median ( X i ). AS: activity score; IM: intermediate metabolizers; NM: normal metabolizers.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques: Clinical Proteomics, Concentration Assay, Activity Assay

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: The cumulative amount-time profile of primaquine (A) and primaquine 5,6-orthoquinone (B) in urine according to the predicted CYP2D6 phenotypes.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques:

Journal: Heliyon

Article Title: An increase in urinary primaquine and a reduction in urinary primaquine-5,6-orthoquinone in the Thai population with CYP2D6 reduced enzyme function

doi: 10.1016/j.heliyon.2024.e24351

Figure Lengend Snippet: The cumulative amount excreted (CAE) in urine of primaquine (A) and primaquine-5,6-orthoquinone (B) and CAE ratio (C) according to the predicted CYP2D6 phenotypes Data are presented as median. p value for the Mann-Whitney U test between normal metabolizers and intermediate metabolizers. CAE: cumulative amount excreted (CAE) in urine; POQ: primaquine-5,6-orthoquinone.

Article Snippet: Two different pairs of primers (Hs00010001_cn and Human RNase P) were used to identify the presence or deletion of the entire CYP2D6 gene versus baseline.

Techniques: MANN-WHITNEY