Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: (A) Myeloma cell lines alone (N=8) were incubated with isotype control antibody or anti-IL-17A mAb to measure proliferation by 3 H-thymidine incorporation after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (B) Myeloma cell lines alone (N=5) were incubated with isotype control antibody or anti-IL-17A mAb to measure metabolic activity by MTT assay after 3 days. Data is presented as percentage inhibition in proliferation in presence of anti-IL-17A mAb compared with isotype control antibody and showed as mean ± SEM. (C) Myeloma cell lines (U266) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. Representative photomicrograph and results (N=3) are presented. Photographs were obtained using a Nikon TE200 microscope (40× objective) with attached camera (Nikon) at room temperature (total magnification 200) and analyzed with Metafluor software (Molecular Devices). The number of colonies were counted in unit area and presented as mean ± SEM. (D) Primary MM cells (N=4) were cultured in methocult agar plates in the presence of isotype control antibody or anti-IL-17A mAb. The number of colonies were counted in unit area and presented as mean ± SEM. *P <0.05. Myeloma cells (U266) was cocultured with BMSC for three days in the presence of isotype, IL-17A with or without anti-IL-6 (10 µg/ml) antibody, and anti-IL-17A antibody with or without IL-6 (10ng/ml) and proliferation was measured by thymidine incorporation (Figure E) and MTT (Figure F) assays. Data is presented as percentage control co-culture proliferation and showed as mean ± SEM.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Incubation, Control, Inhibition, Activity Assay, MTT Assay, Cell Culture, Microscopy, Software, Co-Culture Assay

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: (A) MM cell lines (N=7) were cultured with BMSCs in the presence of isotype control antibody or anti-IL-17A mAb and proliferation measured by 3 H-thymidine incorporation after 3 days and presented as percentage of inhibition in proliferation with isotype control antibody. (B) BMSC was cultured for three days in the presence or absence of IL-17A (100ng/ml), IL-21, IL-22, IL-23, IL-27 and LPS at 10ng/ml concentration. IL-6 production was measured by standard ELISA (R&D Systems, Minneapolis, MN). Bar graph represents mean ± SEM of the data (N=4) calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone without any stimulation. (C) BMSC in the presence (right or absence (left) of MM cell-lines was cultured with isotype control antibody, IL-17A, or anti-IL-17A mAb or in combination with IL-17A + anti-IL-17A mAb and IL-6 levels were measured in culture supernatants by ELISA. Bar graph represents mean ± SEM of the data calculated as percent of increase or decrease in IL-6 levels in culture supernatants compared to IL-6 levels (as endogenous IL-6 production) obtained from BMSC alone with isotype control antibody without any stimulation. (D) Serum-starved MM cells were labeled with calcein AM, washed, and added to BMSC-coated plates in the presence of isotype control antibody or anti-IL-17A mAb for 4 hours and non-adherent cells were removed by washing. Adhesion was measured by measuring the absorbance using 492/520 nm filter set with a fluorescence plate reader. Results represent mean ± SEM of 3 independent experiments performed in triplicate. The absorbance values obtained with isotype control antibody was considered as 100% and percentage of inhibition was calculated for anti-IL-17A treatment. (E) The fetal bone chips with MM cells were incubated in the presence of isotype control antibody or anti-IL-17A mAb for 2 days for ex-vivo for the evaluation of IL-6 production that is measured by ELISA (R & D Systems). Bar graph represents mean ± SEM of the data calculated as percent of IL-6 levels (as endogenous IL-6 production) in culture supernatants from BMSC alone with isotype control antibody. (F) The immunohistochemical analysis of bones was performed by staining them with anti-CD138 antibody. Arrows indicates presence bright CD138+cells. (G) Myeloma cells were co-cultured with BMSC in the presence of IL-17A with or without cell-signaling inhibitors (JAK2, STAT3, JNK, MEK, NFkB and PI3 inhibitors) and IL-6 production was measured with standard ELISA. *P <0.05.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Cell Culture, Control, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Fluorescence, Incubation, Ex Vivo, Immunohistochemical staining, Staining

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: Normal BM cells were cultured for three weeks in osteoclast supporting-medium (consisting 25ng/ml of macrophage colony-stimulating factor and 25 ng/ml of receptor activator of nuclear factor kappa-B ligand) with isotype control antibody (as positive control) or anti-IL-17A antibody and the tartrate-resistant acid phosphatage (TRAP+) multinucleated osteoclast cells were stained and cell-numbers were counted. Cells were cultured without osteoclast-supporting-medium as negative control. Images were obtained at 10 magnifications with microscope (Eclipse TS100, Nikon instruments, Melville, NY, USA) with spot insight camera. TRAP-positive cells containing 5 or more nuclei/cell were enumerated using image J 1.45 software (NIH, Bethesda, MD, USA). A representative image was depicted (Figure A) and composite results from three experiments were shown in bar graph Figure B). Star indicates statistical signifies (p<0.05). There were no significant differences observed among three groups in cell-viability measured with alamor blue staining.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Cell Culture, Control, Positive Control, Staining, Negative Control, Microscopy, Software

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: (A) SCID mice were injected with myeloma cells by sc route. Mice were injected in 2 groups (N=3). One group was injected with myeloma cells with isotype control antibody; and second of mice injected with MM cells in anti-IL-17A mAb (10µg/ml). Tumor volume was measured at indicated time intervals from the MM cell-injections. Representative of two experiments is shown (p<0.05). (B) SCID mice were treated with isotype control antibody or anti-IL-17A mAb (10µg/ml) with weekly sc injections following subcutaneous MM cell-injections (N=4). Tumor volumes (mm 3 ) were measured at indicated time intervals. Representative of two experiments is shown. (C) Balb/C mice were injected with murine plasmacytoma cell-line and treated with isotype control antibody or murine anti-IL-17A mAb (10µg/ml) for a week with sc injections and tumor volumes were measured.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Injection, Control

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: A) SCID mice were transplanted with human fetal bones, and after four weeks, myeloma cells were injected into the bones. One group was treated with vehicle with isotype control antibody and another group of mice were treated subcutaneously with AIN-457 (10µg/ml-/mouse/injection) for four weeks following first detection of tumor by measuring human soluble IL-6R in the serum. Serum samples were collected weekly and level of humans IL-6R was measured by ELISA. Baseline values before treatment were not significantly different among groups. Representative of two experiments is shown (p<0.05). B) Treatment with anti IL-17A protects implanted bones from osteolysis . At the termination of the experiment, implanted human bones were excised and imaged by microcomputed tomography (microCT). Shown are the three-dimensional reconstructions of the bones and sections sliced longitudinally through the midpoint of each specimen injected with MM cells followed by treatment of the animal with either isotype control antibody (excessive bone resorption is seen) or anti IL-17A (no bone resorption is observed, trabecular bone is intact). The areas of increased osteoclastic bone resorption in the isotype control antibody-treated sample are indicated by arrowheads. Scale bar equals 1 mm 3 . Anti-IL-17A antibody reduced the bone resorption caused by myeloma cells. C) Significant differences in the measurements of bone resorption following anti-IL-17A mAb treatment.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Tomography

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: The MM patient samples were collected after informed consent in accordance with the Declaration of Helsinki and approved by the institutional review board (IRB) from Dana-Farber Cancer Institute. Healthy donor bone marrow samples were obtained from AllCells (Emeryville, CA). MM primary cells were purified as described earlier5. RNA was isolated from MM cell-lines (A) and purified MM primary cells (B). Quantitative PCR was performed for IL-17A using 7900HT from Applied Biosystems. Representative experimental results from three different experiments were presented as relative expression value in comparison with GAPDH. For immuno-blot experiments, total cell lysates were prepared from MM cell-lines (C) and purified CD138+ MM primary cells (D), and separated by electrophoresis on 5% to 20% polyacrylamide gradient gels. Samples were probed with anti-sera to IL-17A and GAPDH as indicated. Representative immunoblot of three different experiments was shown. Myeloma cell lines were stained with isotype control antibody or anti–IL-17A antibody and analyzed by confocal microscopy. One representative cell line of 4 experiments was shown at 640 magnifications (E).

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Purification, Isolation, Real-time Polymerase Chain Reaction, Expressing, Comparison, Electrophoresis, Western Blot, Staining, Control, Confocal Microscopy

Journal: Leukemia

Article Title: Targeting IL-17A in Multiple Myeloma: A Potential Novel Therapeutic Approach in Myeloma

doi: 10.1038/leu.2015.228

Figure Lengend Snippet: Myeloma cell-line (RPMI 8226) cells were used to transduce human IL-17A siRNA according to manufactures recommendations to determine the influence on myeloma cell-proliferation and their ability to produce IL-6 when co-cultured with bone-marrow stromal cells. Cell lysates were analyzed using immuno-blots to assess decrease in intra-cellular protein expression of IL-17A (A). Cell-proliferation was analyzed by counting live cells following transfection. Representative study results of three different experiments were shown. MM cell-line (U266) was used to transduce human IL-17A shRNA according to manufactures recommendations to determine the influence on myeloma cell-expression and –proliferation (B). Representative study results of three different experiments were shown. Colony formation was evaluated using MethoCult agar plates following transfection with siRNA or shRNA (C). Representative study results of five different experiments were shown. IL-6 production was measured with standard ELISA following co-culturing myeloma cells with or without transfected MM cells with BMSC (D). Representative study results of three different experiments were shown.

Article Snippet: To evaluate the efficacy of anti-IL-17A mAb in immune competent mice, Balb/C mice were injected with murine plasmacytoma cell-line (ATCC CCL167/MPC11) and treated with isotype control antibody (N=13) or murine anti-IL-17A mAb (N=20) (10μg per mice) for a week and their tumor volumes were measured.

Techniques: Transduction, Cell Culture, Western Blot, Expressing, Transfection, shRNA, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

doi: 10.3389/fimmu.2022.911050

Figure Lengend Snippet: Capacity of cytokine production in memory CD4 + T cell. Cells were activated with PMA/ionomycin for 5 h, adding BFA for the last 3 hours for intracellular cytokine expression. (A) Representative example of cytokine expression in memory CD4 + T cells from young and aged donors after stimulation with PMA/ionomycin. (B) Frequencies in % and geometric mean fluorescence intensity (gMFI) of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TNF-α producing memory CD4 + T cells (mean ± SEM; n = 7).

Article Snippet: Incubation was conducted by dissolving 1x 10 6 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) with 5% human AB serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37˚C in a 5% CO 2 atmosphere Cells were collected, washed, fixed, permeabilized (Inside staining kit, Miltenyi Biotec) and stained with a combination of monoclonal antibodies including anti-human-IFNγ (PERCP-Cy5.5, Biolegend, clone 4SB3, Cat.# 502526), anti-human-IL-2 (APC-vio770, Miltenyi Biotec, clone N7.48 A, Cat.# 130-097-011), anti-human-IL-4 (PE, Miltenyi Biotec, clone 7A3-3, Cat.# 130-091-647), anti-human-IL-17A (FITC, Miltenyi Biotec, clone CZ8-23G1, Cat.# 130-094-520), and anti-human-TNF-α (PE-Vio770 (Miltenyi Biotec, clone cA2, Cat.# 130-096-755).

Techniques: Expressing, Fluorescence

Journal: PLOS ONE

Article Title: Anti-inflammatory effect of the combined treatment of LMT-28 and kaempferol in a collagen-induced arthritis mouse model

doi: 10.1371/journal.pone.0302119

Figure Lengend Snippet: (A) Arthritis scores of mice were measured every 3 days starting at 21 days after the first immunization. (B) DBA1/J mice showed deformation and swelling of the toe and ankle joints. (C) Paraffin-embedded sections of CIA mice joint were stained with hematoxylin and eosin. (D) Histological analysis was performed based on the analysis criteria: inflammation, synovial hyperplasia, pannus formation, and erosion of cartilage and bone. (E) The protein levels of IL-6, IL-1β and IL-17 in CIA mouse serum were measured using ELISA. Data are presented as mean ± SEM. # p <0.05 vs. normal group, * p <0.05, ** p <0.01, and *** p <0.005 vs. CIA control group.

Article Snippet: The cells were incubated with anti-IL-17A-PE antibody (Miltenyi Biotec) or anti-mouse FOXP3 antibody (Invitrogen, Waltham, MA, USA) diluted in 1× permeabilization buffer (eBioscience) for 30 min at 4°C in the dark.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Veterinary Science

Article Title: Intramammary Immunisation Provides Short Term Protection Against Mannheimia haemolytica Mastitis in Sheep

doi: 10.3389/fvets.2021.659803

Figure Lengend Snippet: Details of antibodies used for cytokine ELISAs.

Article Snippet: Bovine IL-17A Coating , Polyclonal , n/a, rabbit , 2 , Kingfisher Biotech.

Techniques: