Journal: Oncology Letters

Article Title: Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells

doi: 10.3892/ol.2016.5285

Figure Lengend Snippet: bDLE increases the expression of CD42a + megakaryocytic marker differentiation in K562 cells. (A) Untreated cells, (B) 0.07 U/ml bDLE, (C) 0.14 U/ml bDLE, (D) 0.21 U/ml bDLE, (E) 0.28 U/ml bDLE, (F) 0.35 U/ml bDLE, (G) 10 ng/ml phorbol myristate acetate or (H) dimethyl sulfoxide (1.5%, v:v) were incubated for 96 h. Cells were harvested and incubated with peridinin chlorophyll protein complex conjugated-anti-CD42a in PBS with 1% fetal bovine serum and 0.1% sodium azide for 30 min at 4°C. Samples were washed and resuspended in PBS, and 10,000 events were analyzed by flow cytometry. Flow cytometry data show representative results from one of three independent experiments. CD, cluster of differentiation; bDLE, bovine dialyzable leukocyte extract.

Article Snippet: Cell viability assessment by PI staining To evaluate cell death, cells were centrifuged at 500 × g at 25°C for 10 min and washed with PBS, resuspended in 0.2 ml PBS and PI (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 min at room temperature (25°C) in the dark, followed by analysis with an Accuri™ C6 flow cytometer (BD Biosciences).

Techniques: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

Journal: Oncology Letters

Article Title: Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells

doi: 10.3892/ol.2016.5285

Figure Lengend Snippet: bDLE inhibits the cell proliferation rate of K562 cells. K562 cells were treated with or without bDLE, PMA or DMSO and incubated for 96 h. Following the incubation, the cells were harvested and resuspended in PBS and the cell proliferation percentage was estimated by cell counting for each treatment group by trypan blue staining. *P

Article Snippet: Cell viability assessment by PI staining To evaluate cell death, cells were centrifuged at 500 × g at 25°C for 10 min and washed with PBS, resuspended in 0.2 ml PBS and PI (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 min at room temperature (25°C) in the dark, followed by analysis with an Accuri™ C6 flow cytometer (BD Biosciences).

Techniques: Incubation, Cell Counting, Staining

Journal: Oncology Letters

Article Title: Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells

doi: 10.3892/ol.2016.5285

Figure Lengend Snippet: bDLE induces macrophage polarization to M2 assessed by expression of the CD163 + marker in K562 cells. (A) Untreated cells, (B) 0.07 U/ml bDLE, (C) 0.14 U/ml bDLE, (D) 0.21 U/ml bDLE, (E) 0.28 U/ml bDLE, (F) 0.35 U/ml bDLE, (G) 10 ng/ml phorbol myristate acetate or (H) dimethyl sulfoxide (1.5%, v:v) were incubated for 96 h. Cells were harvested and incubated with anti-CD68 and anti-CD163 in PBS with 1% fetal bovine serum and 0.1% sodium azide for 30 min at 4°C. Samples were washed and resuspended in PBS, and 10,000 events were analyzed by flow cytometry. CD, cluster of differentiation; bDLE, bovine dialyzable leukocyte extract.

Article Snippet: Cell viability assessment by PI staining To evaluate cell death, cells were centrifuged at 500 × g at 25°C for 10 min and washed with PBS, resuspended in 0.2 ml PBS and PI (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 min at room temperature (25°C) in the dark, followed by analysis with an Accuri™ C6 flow cytometer (BD Biosciences).

Techniques: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

Journal: Oncology Letters

Article Title: Effect of bovine dialyzable leukocyte extract on induction of cell differentiation and death in K562 human chronic myelogenous leukemia cells

doi: 10.3892/ol.2016.5285

Figure Lengend Snippet: bDLE induces monocytic differentiation in K562 cells assessed by the expression of the CD14 + surface marker. (A) Untreated cells, (B) 0.07 U/ml bDLE, (C) 0.14 U/ml bDLE, (D) 0.21 U/ml bDLE, (E) 0.28 U/ml bDLE, (F) 0.35 U/ml bDLE, (G) 10 ng/ml phorbol myristate acetate or (H) dimethyl sulfoxide (1.5%, v:v) were incubated for 96 h. Cells were harvested and incubated with anti-CD14 phycoerythrin Texas red in PBS with 1% fetal bovine serum and 0.1% sodium azide for 30 min at 4°C. Samples were washed and resuspended in PBS, and 10,000 events were analyzed by flow cytometry. Flow cytometry data shows representative results from one of three independent experiments. CD, cluster of differentiation; bDLE, bovine dialyzable leukocyte extract.

Article Snippet: Cell viability assessment by PI staining To evaluate cell death, cells were centrifuged at 500 × g at 25°C for 10 min and washed with PBS, resuspended in 0.2 ml PBS and PI (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 5 min at room temperature (25°C) in the dark, followed by analysis with an Accuri™ C6 flow cytometer (BD Biosciences).

Techniques: Expressing, Marker, Incubation, Flow Cytometry, Cytometry

Journal: Tissue Engineering. Part A

Article Title: Secreted Frizzled Related Protein 4 Reduces Fibrosis Scar Size and Ameliorates Cardiac Function After Ischemic Injury

doi: 10.1089/ten.tea.2009.0739

Figure Lengend Snippet: Administration of Sfrp4 interferes with Wnt canonical signaling. ( A ) Ischemic hearts treated by IM injection of PBS or 20 μg Sfrp4 protein were sectioned 10 weeks after LAD ligation. Slides were costained with antibodies against collagen type III (green) and cTn-T (red). The extent of the noncardiac acellular scar (lack of cTn-T and 4′,6-diamidino-2-phenylindole (DAPI) staining), indicated by white arrows, was suppressed by administration of Sfrp4. ( B–D ) About 100 μL PBS or 20 μg Sfrp4 protein was injected into ischemic border areas just after LAD ligation to investigate the effects on Wnt canonical signaling at early time points. Heart sections 3 days after LAD ligation were stained for β-catenin (green, B ), Sfrp4 (red, B ), active β-catenin (green, C ), and inactive serine 9 phosphorylated glycogen syntase kinase (GSK)-3β (red, D ). DAPI (blue) was used to observe nuclei and hence cellular versus acellular areas. The border between the acellular scar and cellular areas is shown by dotted white lines. The average of total fibrous tissue areas (in mm 2 ) detected by Masson's Trichrome staining in adjacent sections is indicated with standard deviations ( C, D ).

Article Snippet: Five or 20 μg recombinant human Sfrp4 (sFRP-4; R & D Systems, Inc.) in 50 μL phosphate-buffered saline (PBS) was mixed with 50 μL collagen type I gel (BD Biosciences) and injected to the ischemic border zones.

Techniques: Injection, Ligation, Staining

Journal: Tissue Engineering. Part A

Article Title: Secreted Frizzled Related Protein 4 Reduces Fibrosis Scar Size and Ameliorates Cardiac Function After Ischemic Injury

doi: 10.1089/ten.tea.2009.0739

Figure Lengend Snippet: Histology of PBS- or Sfrp4-treated hearts. Adult normal rat heart ( A ) and ischemic hearts ( B–H ) generated by LAD ligation were sectioned and stained by Masson's Trichrome 10 weeks after LAD ligation. ( B ) Twenty-microgram Sfrp4 protein-injected heart, ( C ) 2.5 × 10 6 cubes of S-PH injected heart, ( D ) 5 × 10 6 cubes of S-PH immobilized collagen sheet-applied heart, ( E ) nontreated ischemic heart section, ( F ) PBS-injected heart, ( G ) 2.5 × 10 6 cubes of empty polyhedra-injected heart, ( H ) 5 × 10 6 cubes of empty polyhedra-immobilized collagen sheet-applied heart. Photo of representative cross section of each group ( n = 4) is shown. ( I ) Means of fibrosis percentage and wall thickness for individual treatment groups plotted against each other. Vertical and horizontal lines indicate standard errors of mean measurements for fibrosis and wall thickness, respectively; the diagonal line indicates the line of best fit. IM injection of PBS or Sfrp4; PH IM injection of empty or S-PH; PH-sheet, application of empty or S-PH immobilized on collagen sheet; PH-glue, empty or S-PH applied with fibrin glue. Filled circles indicate control treatments; empty circles indicate Sfrp4-containing treatments. Sfrp4 treatments result in hearts with significantly less fibrosis and thicker walls ( p = 8.4e-4) as indicated by multivariate ANOVA.

Article Snippet: Five or 20 μg recombinant human Sfrp4 (sFRP-4; R & D Systems, Inc.) in 50 μL phosphate-buffered saline (PBS) was mixed with 50 μL collagen type I gel (BD Biosciences) and injected to the ischemic border zones.

Techniques: Generated, Ligation, Staining, Injection

Journal: Tissue Engineering. Part A

Article Title: Secreted Frizzled Related Protein 4 Reduces Fibrosis Scar Size and Ameliorates Cardiac Function After Ischemic Injury

doi: 10.1089/ten.tea.2009.0739

Figure Lengend Snippet: . ( A ) IM injection of Sfrp4 in an acute ischemic model. Ischemic hearts were generated by LAD ligation, and 100 μL of PBS ( n = 7), 5 μg Sfrp4 protein ( n = 6), or 20 μg Sfrp4 protein ( n = 9) was injected to the ischemic border zones soon after LAD ligation. ( B ) IM injection of Sfrp4 in a subacute ischemic model. About 100 μL of PBS ( n = 5) or 20 μg Sfrp4 protein ( n = 6) was administered intramuscularly 2 weeks after LAD ligation. ( C, D ) IM injection of Sfrp4 just after ( C ) or before ( D ) recanalization injury. About 100 μL PBS or 20 μg Sfrp4 protein was administered intramuscularly after or before a transient 1 h LAD ligation. All treatments except 5 μg Sfrp4 ( A ) show either significant ( p

Article Snippet: Five or 20 μg recombinant human Sfrp4 (sFRP-4; R & D Systems, Inc.) in 50 μL phosphate-buffered saline (PBS) was mixed with 50 μL collagen type I gel (BD Biosciences) and injected to the ischemic border zones.

Techniques: Injection, Generated, Ligation

Journal: Tissue Engineering. Part A

Article Title: Secreted Frizzled Related Protein 4 Reduces Fibrosis Scar Size and Ameliorates Cardiac Function After Ischemic Injury

doi: 10.1089/ten.tea.2009.0739

Figure Lengend Snippet: Administration of Sfrp4 in a slow-releasing cube polyhedra form demonstrated long-term therapeutic effects. ( A ) Schema for generation of empty polyhedra and Sfrp4 immobilized in polyhedra (S-PH) and scanning electron microscopic image of S-PH. ( B ) Concentration of Sfrp4 in the culture medium after culture of primary rat cardiomyocytes in the presence of S-PH ( n = 3) for 2 or 4 days as determined by ELISA. Mean values and standard deviations are indicated. ( C ) Twenty microliter PBS ( n = 5), 2.5 × 10 6 cubes of empty polyhedra ( n = 6), 20 μg Sfrp4 protein ( n = 5), or 2.5 × 10 6 cubes of S-PH ( n = 6) were administered just after LAD ligation. EFs of indicated groups are shown. The full data set contains significant treatment effects as determined by repeated measures ANOVA ( p = 1.1e-4). Significant ( p = 0.016) time–treatment interaction effects were also observed between polyhedra bound and soluble Sfrp4. Complete set of p .

Article Snippet: Five or 20 μg recombinant human Sfrp4 (sFRP-4; R & D Systems, Inc.) in 50 μL phosphate-buffered saline (PBS) was mixed with 50 μL collagen type I gel (BD Biosciences) and injected to the ischemic border zones.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Ligation

Journal: Tissue Engineering. Part A

Article Title: Secreted Frizzled Related Protein 4 Reduces Fibrosis Scar Size and Ameliorates Cardiac Function After Ischemic Injury

doi: 10.1089/ten.tea.2009.0739

Figure Lengend Snippet: Sfrp4 suppresses cell proliferation and collagen production after ischemia. ( A ) BrdU incorporation during day 3 to 4 after LAD ligation. About 2.5 × 10 6 cubes of empty polyhedra (empty-PH), or 2.5 × 10 6 cubes of S-PH were injected into ischemic border areas just after LAD ligation. BrdU was administered at day 3. The border between the acellular scar and cellular areas is shown by dotted black lines. Cubes visible in the bottom of the photo are S-PH in the ischemic border area. The mean and standard deviations of the number of BrdU-positive cells are indicated in the figure. ( B ) Vascular density in the ischemic border area of PBS- or Sfrp4-treated hearts. The number of von Willebrand Factor-expressing vessel-like structures in border areas of PBS- or Sfrp4-treated rats 3 days after LAD ligation. Means and standard deviations of vascular density (vessels per mm 2 ) are shown in the figure. Red arrows indicate von Willebrand Factor-positive vessel-like structures. Black line framed area in upper photo is magnified and shown (indicated as block arrow) in lower photo. ( C ) Transcript levels of the β-catenin effector gene TCF4 , the TCF4 target cyclin D2 , and collagen IIIa (Col IIIa) from PBS or 20 μg Sfrp4-treated ischemic border areas 3 days after ischemic heart injury as determined by qRT-PCR. Values were normalized by the expression of glyceraldehyde 3-phosphate dehydrogenase (rat glyceraldehyde 3-phosphate dehydrogenase as 10,000). The bar and error bar show mean and standard deviations of three rat heart samples, respectively. p -Values for differences in mean values for PBS- or Sfrp4-treated tissues are indicated in the figure.

Article Snippet: Five or 20 μg recombinant human Sfrp4 (sFRP-4; R & D Systems, Inc.) in 50 μL phosphate-buffered saline (PBS) was mixed with 50 μL collagen type I gel (BD Biosciences) and injected to the ischemic border zones.

Techniques: BrdU Incorporation Assay, Ligation, Injection, Expressing, Blocking Assay, Quantitative RT-PCR

Journal: Cytokine

Article Title: IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis

doi: 10.1016/j.cyto.2018.07.034

Figure Lengend Snippet: IL-36γ treatment induces the transient production of immune mediators in murine lower FRT tissue and vaginal lavages. (A) Female reproductive tract tissue was removed from C57Bl/6 mice 4h or 24h after i.vag. IL-36γ treatment (250 ng) and the lower FRT was isolated for RNA extraction ( n = 5/time point). Expression of genes was measured by qRT-PCR and was normalized relative to Gapdh . Data is representative of two independent animal experiments and represent mean fold change ± SD relative to PBS treated controls. (B) Female C57Bl/6 mice were treated with murine recombinant IL-36γ (250 ng) or PBS. Vaginal lavages ( n = 5 mice per time point) were collected at 4, 24 and 48h after exposure. Levels of the chemokines; CCL20, IP-10, and KC were quantified by cytometric bead array analysis. Minimum detectable concentration is indicated by dashed line. Data shown are representative of two independent animal experiments and are presented as the mean ± SD. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple comparisons test. *, P

Article Snippet: Mice were i.vag. treated by instilling recombinant murine IL-36γ (100 ng, 250 ng, or 500 ng; BioLegend) or PBS (Corning, Manassas, VA) in 10 μl total volume.

Techniques: Mouse Assay, Isolation, RNA Extraction, Expressing, Quantitative RT-PCR, Recombinant, Concentration Assay

Journal: Cytokine

Article Title: IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis

doi: 10.1016/j.cyto.2018.07.034

Figure Lengend Snippet: IL-36γ transiently promotes polymorphonuclear leukocyte infiltration in the vaginal microenvironment. Female C57Bl/6 mice were treated with murine recombinant IL-36γ (250 ng) or PBS and vaginal swabs were collected 4h (A, B, C) and 24h (data not shown) after treatment ( n = 5 mice/treatment). Vaginal smears were prepared on slides and allowed to air dry. Slides were stained with modified Wright stain and imaged at 20× and 40× magnification. Black arrows indicate polymorphonuclear leukocytes (PMN) and white arrows indicate epithelial cells. Scale bar is 50 μm. Cells in fields at 20× were enumerated and graphed as average number of total cells/mm 2 (D), average number of epithelial cells/mm 2 (E), and average number of PMN/mm 2 (F). Statistical significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test. ***, P

Article Snippet: Mice were i.vag. treated by instilling recombinant murine IL-36γ (100 ng, 250 ng, or 500 ng; BioLegend) or PBS (Corning, Manassas, VA) in 10 μl total volume.

Techniques: Mouse Assay, Recombinant, Staining, Modification, Wright Stain

Journal: Cytokine

Article Title: IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis

doi: 10.1016/j.cyto.2018.07.034

Figure Lengend Snippet: IL-36γ treatment 4h prior to infection significantly protects against HSV-2 disease incidence, reduces disease severity, and enhances survival. Female six- to eight-week-old C57Bl/6 mice were treated with murine recombinant IL-36γ (250 ng) 4h prior to infection ( n =10), or mock-treated with PBS ( n = 10). Mice were then intravaginally challenged with a lethal dose of HSV-2 186 (10 3 PFU). (A) Vaginal swabs were collected at days 2 and 3 post-inoculation and HSV-2 replication was measured in duplicate by standard plaque assay. Each symbol represents an individual mouse, and mean ± SD is depicted. Dashed line represents minimum detectable level for assay. Several IL-36γ-treated mice had undetectable titers and are depicted on the graph as half of the minimum detectable level. Disease severity (B) was measured daily and scored on a 0–5 scale. Once a mouse scored 5 and died it was no longer included in scoring. Incidence of disease was measured by the presence of both erythema and hair loss (C), and survival (D) was recorded over a 21-day period. Data is representative of two independent animal studies. Statistical analyses were performed by one-way ANOVA with Bonferroni’s multiple comparisons test (A), Area Under Curve (AUC) analysis with an unpaired two-tailed Student t -test with Welch’s correction (B), and log-rank analysis (C, D). **, P

Article Snippet: Mice were i.vag. treated by instilling recombinant murine IL-36γ (100 ng, 250 ng, or 500 ng; BioLegend) or PBS (Corning, Manassas, VA) in 10 μl total volume.

Techniques: Infection, Mouse Assay, Recombinant, Plaque Assay, Two Tailed Test